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Protein synthesis and myosin heavy chain mRNA in the rat diaphragm during mechanical ventilation

University of Florida Institutional Repository

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PROTEIN SYNTHESIS AND MYOSIN HEAVY CHAIN mRNA IN THE RAT DIAPHRAGM DURING MECHANICAL VENTILATION By R. ANDREW SHANELY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2002

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Copyright 2002 by R. Andrew Shanely

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The completion of this Dissertation is one my greatest accomplishments and is dedicated to my parents. If it were not for your support and enthusiasm of my educational development I would never have contemplated postgraduate work, let alone completed it. Each of you has given me the love, understanding, encouragement, and freedom to lead a happy and successful life. Thank you. This Dissertation is also dedicated to Shannon. Your love, support, and daily encouragement never ceases to amaze me. My love, thank you for your day-to-day help and understanding throughout my postgraduate career.

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ACKNOWLEDGMENTS I would like to acknowledge those who made the successful completion of this project possible: my mentor, Dr. Scott Powers; and my committee members, Dr. Stephen Dodd, Dr. Randy Braith, and Dr. Paul Davenport. Collectively and individually, my committee provided me with invaluable guidance, instruction, and patience. I would also like to acknowledge Darin Van Gammeren, Michael McKenzie, and Murat Zergeroglu for their contribution to this project, without which these experiments would not have been possible. Kevin Yarasheskis measurement of [ 13 C]leucine incorporation into diaphragmatic proteins was the crux of these experiments. His enthusiastic participation assured the success of these experiments. Fadia Hadads knowledge and patience made the measurement of myosin heavy chain mRNA a reality. Jeff Coombes, Haydar Demirel, and Hisashi Natio must be acknowledged for their immediate guidance upon my arrival in Dr. Scott Powers laboratory and thus their indirect contribution to this project. I am forever indebted to Dr. Powers for serving as my mentor. His motivation, guidance, support, mentorship, and friendship have been unflagging. A student is only capable of what his mentor demands! This work was made possible by funding from the National Institutes of Health (HL-62361). iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABSTRACT.........................................................................................................................x CHAPTER 1 INTRODUCTION............................................................................................................1 Objectives of Specific Aim #1........................................................................................2 Aim #1: Rationale for Experimental Approach and Hypothesis....................................2 Objectives of Specific Aim #2........................................................................................3 Aim #2: Rationale for Experimental Approach and Hypothesis....................................3 2 LITERATURE REVIEW.................................................................................................4 History of Mechanical Ventilation..................................................................................4 Indication for Clinical Use of Mechanical Ventilation...................................................6 Modes of Mechanical Ventilator Operation...................................................................8 Controlled Mechanical Ventilation..........................................................................8 Assist-Control Ventilation.......................................................................................9 Intermittent Mandatory Ventilation.........................................................................9 Pressure Support Ventilation.................................................................................10 Diaphragmatic Motion during Mechanical Ventilation................................................10 Weaning from Mechanical Ventilation.........................................................................12 Properties of the Diaphragm.........................................................................................14 Function of the Diaphragm...........................................................................................14 Metabolic Characteristics of the Diaphragm................................................................15 Skeletal Muscle Fiber Types within the Diaphragm..............................................16 Oxidative Capacity.................................................................................................18 Muscle Atrophy............................................................................................................19 Models of Locomotor Muscle Atrophy........................................................................20 Reduced Electrical Activation and Load Bearing..................................................20 Reduced Loading...................................................................................................22 Models of Inactivity...............................................................................................24 v

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Models of Atrophy in the Diaphragm...........................................................................26 Procedure for Investigating Diaphragmatic Atrophy.............................................27 Electromyographic Activity...................................................................................27 Diaphragmatic Mass...............................................................................................28 Differential Fiber Type Response to Diaphragmatic Inactivity.............................28 Myosin Heavy Chain Content................................................................................29 Myosin Heavy Chain mRNA.................................................................................29 Contractile Properties.............................................................................................30 Oxidative Capacity.................................................................................................30 Protein Synthesis....................................................................................................30 Diaphragm Length Changes...................................................................................31 Mechanical Ventilation and Diaphragmatic Atrophy...................................................32 Electrical Activity..................................................................................................32 Diaphragmatic Disuse Atrophy..............................................................................32 Animal Models and Mechanical Ventilation.........................................................33 Effects of Mechanical Ventilation on Diaphragmatic Contractile Properties........34 Mechanical Ventilation and Diaphragmatic Atrophy............................................35 Summary of Literature Review.....................................................................................37 3 METHODS.....................................................................................................................40 Experimental Design-Specific Aim #1.........................................................................40 Animals and Experimental Design.........................................................................40 Mechanical ventilation protocol......................................................................41 Postmortem examination.................................................................................43 Control animals (nonmechanically ventilated) protocol..................................43 Methods Used: Biochemical Assays......................................................................44 Tissue removal and storage..............................................................................44 Rates of in vivo diaphragmatic protein synthesis.............................................44 Statistical analysis............................................................................................49 Experimental Design-Specific Aim #2.........................................................................49 Methods Used: Biochemical Assays......................................................................49 Total RNA isolation.........................................................................................49 Reverse transcription (RT)...............................................................................50 Polymerase chain reaction (PCR)....................................................................51 Analysis of gels................................................................................................52 Statistical analysis............................................................................................53 4 RESULTS.......................................................................................................................54 Morphological, Physiological, and Post Mortem Observations...................................54 Influence of Mechanical Ventilation on Protein Synthesis...........................................55 Total RNA and Myosin Heavy Chain mRNA in the Diaphragm after Spontaneous Breathing and Mechanical Ventilation....................................................................57 vi

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5 DISCUSSION.................................................................................................................71 Overview of Principle Findings....................................................................................71 Impact of Mechanical Ventilation on Protein Synthesis in the Diaphragm..................71 Mixed Muscle Protein Synthesis............................................................................71 Myosin Heavy Chain Protein Synthesis.................................................................72 Myosin Heavy Chain mRNA.................................................................................74 Regulation of Protein Synthesis....................................................................................75 Transcription..........................................................................................................75 Translation Initiation........................................................................................76 Translation Elongation and Termination.........................................................78 Critique of the Experimental Model.............................................................................79 Nutritional Status...................................................................................................80 Anesthesia..............................................................................................................80 Infusion period.......................................................................................................81 Blood Removal......................................................................................................82 Mode of Mechanical Ventilation...........................................................................83 Summary and Future Experiments................................................................................83 LIST OF REFERENCES...................................................................................................85 BIOGRAPHICAL SKETCH...........................................................................................103 vii

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LIST OF TABLES Table page 2-1 Fiber type composition (%) of the diaphragm and locomotor skeletal muscles.........17 2-2 Bioenergetic enzyme activities in the costal diaphragm and two locomotor muscles...................................................................................................................19 3-1 Oligonucleotide primers used for the PCR reactions..................................................53 4-1 Animal body mass before and after experimental period............................................58 4-2 Heart rate response during MV and SB.......................................................................59 4-3 Systolic blood pressure response during MV and SB.................................................59 4-4 Fractional synthetic rates of mixed muscle protein and myosin heavy chain protein by calculation with each surrogate of the [ 13 C]leucyl-tRNA precursor pool........................................................................................................................65 4-5 Total RNA obtained from the costal diaphragm.........................................................66 viii

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LIST OF FIGURES Figure page 3-1 Experimental design for Specific Aim #1...................................................................41 3-2 Schematic representation of the myosin heavy chain (MHC) genes...........................53 4-1 Plasma [ 13 C]leucine and plasma [ 13 C]ketoisocaproic acid ([ 13 C]KIC) enrichment....60 4-2 Tissue fluid [ 13 C]leucine enrichment in the diaphragm...............................................61 4-3 Mixed muscle protein and myosin heavy chain [ 13 C]leucine enrichment in the diaphragm..............................................................................................................62 4-4 Fractional synthetic rates of mixed muscle protein (MMP) by calculation with tissue fluid [ 13 C]leucine.........................................................................................63 4-5 Fractional synthetic rates of myosin heavy chain (MHC) protein by calculation with tissue fluid [ 13 C]leucine.................................................................................64 4-6 RT-PCR products........................................................................................................67 4-7 Relative type I MHC expression.................................................................................67 4-8 Relative type IIa MHC expression..............................................................................68 4-9 Relative type IIx MHC expression..............................................................................69 4-10 Relative type IIb MHC expression............................................................................70 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy PROTEIN SYNTHESIS AND MYOSIN HEAVY CHAIN mRNA IN THE RAT DIAPHRAGM DURING MECHANICAL VENTILATION By R. Andrew Shanely December 2002 Chair: Scotty K. Powers, Ph.D., Ed.D. Department: Exercise and Sport Sciences The purpose of these experiments was to test the hypothesis that mechanical ventilation-induced diaphragmatic atrophy is due, at least in part, to a decreased rate of total protein synthesis and myosin heavy chain (MHC) protein synthesis. We also tested the hypothesis that mechanical ventilation (MV) alters pretranslational events in the diaphragm. To test these hypotheses, we randomly assigned specific-pathogenfree barrier-protected 4-month-old female Sprague-Dawley rats to one of three experimental groups: MV; spontaneously breathing (SB); or control/acute anesthesia. The MV animals were mechanically ventilated for 6, 12, or 18 hours (n=10 for each time period). Spontaneously breathing animals underwent the same surgical procedures and were anesthetized for the same period of time as the animals in the MV group, but were not exposed to MV. The acute-control animals (n=10) were not exposed to MV or prolonged anesthesia. The rate of protein synthesis was determined by measuring the rate of [ 13 C]leucine incorporation into total protein and MHC protein in the diaphragm of the x

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MV and SB rats. We isolated total RNA from the diaphragms of all groups and measured the expression of type I, IIa, IIx, and IIb (the four adult MHC mRNA isoforms). The rate of protein synthesis of the MV rats was compared to that of the SB rats at the 6, 12, and 18 hour time points. Six hours of MV caused a significant decrease (30%, p < 0.05) in the rate of total protein synthesis and a significant decrease (65%, p < 0.05) in the rate of MHC protein synthesis. The decrease (p < 0.05) in protein synthesis remained at this depressed level after 12 and 18 hours of MV. Expression of MHC mRNA isoforms in the diaphragms of MV animals and the SB animals did not change (p > 0.05). These data support the hypothesis that a decrease in protein synthesis contributes to MV-induced diaphragmatic atrophy. In contrast, these data do not support the hypothesis that MV alters pretranslational events in the diaphragm. xi

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CHAPTER 1 INTRODUCTION Mechanical ventilation (MV) provides a means of supporting blood gas homeostasis for patients who cannot maintain adequate alveolar ventilation. Unfortunately, prolonged MV (i.e., 3 days) is not without consequence because as many as 20% of patients experience difficulty in weaning from the ventilator (94). While the underlying cause for weaning difficulties has yet to be fully elucidated, respiratory muscle atrophy and the associated contractile dysfunction are potential mechanisms (167). In this regard, Anzueto et al. (8) reported significant reductions in diaphragmatic strength and endurance of healthy baboons after 11 days of MV. Despite these important physiological findings, Anzueto and collegues report did not investigate biochemical or histological alterations in the diaphragm associated with MV. Further, Le Bourdelles et al. (92) examined the effects of 48 hours of controlled MV on both atrophy and contractile properties in the rat diaphragm. They reported a significant reduction in isometric force generation and a reduction in both diaphragmatic mass (i.e., atrophy) and protein content (92). Powers et al. (125) have also reported that MV leads to progressive diaphragmatic contractile dysfunction. These experiments demonstrated a significant correlation between time on the ventilator and contractile dysfunction (i.e., the greater the time on the ventilator the greater the degree of diaphragmatic contractile dysfunction) (125). Finally, recent experiments in our laboratory have demonstrated that MV for little as 18 hours results in diaphragmatic atrophy (145). Preliminary experiments in our 1

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2 laboratory suggest that the observed diaphragmatic atrophy is associated with a decreased rate of diaphragmatic protein synthesis and a decrease in myosin heavy chain (MHC) content. These observations form the basis for the proposed experiments. Objectives of Specific Aim #1 The effect of MV on diaphragmatic protein synthesis was determined. We tested the hypothesis that MV-induced diaphragmatic atrophy is due, at least in part, to a decreased rate of total and myofibrillar protein synthesis. Aim #1: Rationale for Experimental Approach and Hypothesis Preliminary experiments in our laboratory indicated that prolonged MV results in significant diaphragmatic atrophy. The extent to which decreases in protein synthesis contribute to the MV-induced loss of diaphragmatic contractile protein is unknown. Therefore, these experiments were designed to determine the time course of changes in protein synthesis during 6, 12, and 18 hours of MV. This was achieved by measuring both total and contractile protein synthesis rates (in vivo) in the diaphragms of control and MV animals. Specifically, diaphragmatic protein synthesis was measured over the course of the last 6 hours of the experimental period (e.g., 12 to18 hours of MV). The fractional rate of diaphragm muscle protein synthesis was measured using intravenous infusion of [113 C]leucine. We quantified the in vivo rate of incorporation of [113 C]leucine into both total and contractile proteins in the diaphragm by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The stable isotope [113 C]leucine was chosen for several reasons: Less isotope effect (i.e., less tissue injury). Small tissue sample is required for analysis. Safety.

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3 Validity and reliability of this label for measurement of the rate of in vivo muscle protein synthesis is well established (121). Objectives of Specific Aim #2 The effect of MV on diaphragmatic myosin heavy chain (MHC) mRNA content was determined. We tested the hypothesis that MV alters pretranslational events in the diaphragm. Aim #2: Rationale for Experimental Approach and Hypothesis Preliminary experiments suggested that prolonged MV results in a decrease in diaphragmatic protein synthesis. The extent to which pretranslational events contribute to the MV-induced decrease in protein synthesis is unknown. Therefore, these experiments were designed to determine the time course of changes in MHC mRNA after 6, 12, and 18 hours of MV. This was achieved by measuring the MHC mRNA content in diaphragms from control and MV animals. Specifically, we measured the diaphragmatic content of type I, IIa, IId/x, and IIb MHC mRNA in control animals and at the completion of 6, 12, and 18 hours of MV.

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CHAPTER 2 LITERATURE REVIEW Mechanical ventilation (MV) is an intervention used to sustain ventilation in patients who are unable to maintain adequate alveolar ventilation. The withdrawal of MV is commonly referred to as weaning. Patients who experience weaning difficulties commonly exhibit respiratory muscle weakness. Hence, it has been postulated that both weakness and decreased endurance of respiratory muscles are major contributors to the failure to wean patients from MV (167). This notion is strongly supported by recent animal studies indicating that prolonged controlled MV results in significant reductions in diaphragmatic force production. Further, our laboratory recently discovered that the MV-induced diaphragmatic force deficit is associated with significant diaphragmatic. History of Mechanical Ventilation Galen (56), in the year AD 160, may have been the first to artificially ventilate an animal. He reported that If you take a dead animal and blow air through its larynx (through a reed), you will fill its bronchi and watch its lungs attain the greatest distention. More than 1000 years after Galen, Vesalius found that he could keep the heart beating after a pneumothorax by inflating the lungs through a reed tied to the trachea (171). In 1664 Hooke described dissecting a dog, putting a pipe into the trachea and attaching the pipe to a bellows (15). The heart continued beating and the dog stayed alive for over an hour (15). Artificial ventilation of dogs led to the use of positive pressure ventilation to revive human drowning victims in the mid 1700s (35). However, positive pressure ventilation 4

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5 frequently caused fatal pneumothoraxes during animal experiments and was later condemned by both the Academie Francaise and the Royal Humane Society (35). Quashing positive pressure ventilation early in its development led to an alternative method: negative pressure ventilation. Development and use of negative pressure ventilation flourished in the 1800s and by 1928 the Iron Lung became the first negative pressure ventilator used successfully on a large scale (35). The iron lung saved many lives during the poliomyelitis epidemic in the 1930s and served as the mainstay of treatment for respiratory paralysis from poliomyelitis until positive pressure ventilation was reintroduced in the 1950s (35). Positive pressure ventilation was heavily used in physiology laboratories during the mid to late 1800s and by 1879 the volume-cycled ventilator was a common piece of equipment at Harvard University (35). While positive pressure ventilation was used to some degree before the 1950s, it was not until the poliomyelitis epidemic struck Copenhagen in 1952 that its full utility was realized (35). Since then, positive pressure ventilation has been used successfully to treat many medical conditions that lead to respiratory insufficiency. In the 1980s a new method of positive pressure ventilation was introduced. This new application was a noninvasive means of ventilation via a nasal, facemask, or an oral connection and proved to be a significant development in MV (133). The evolution of the mechanical ventilator has continued to this day. Its use as an indispensable life-saving tool has insured its place in clinical practice.

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6 Indication for Clinical Use of Mechanical Ventilation MV is used for 4 main reasons: Life support for a patient with a life threatening illness whose recovery is anticipated. Life support for a patient under general anesthesia during surgery. To provide ventilation during respiratory muscle failure or to compensate for a damaged upper airway. As an aid during recovery or rehabilitation from an illness (3). A common physiological outcome of these situations is respiratory failure. Respiratory failure is often defined as a PaO 2 of less than 50 mmHg at sea level while breathing a gas mixture of at least 50% O 2 and/or a PaCO 2 greater than 50 mmHg (hypercapnia) (3). Respiratory failure due to inadequate gas exchange is termed hypoxic respiratory failure (134). If respiratory failure is due to ventilatory pump failure it is known as hypercapnic respiratory failure, or the two may occur in combination (134). Hypoxic respiratory failure is commonly associated with severe respiratory illnesses and can induce hypoxemia by one or some combination of four mechanisms: alveolar hypoventilation, right-to-left shunt in the heart, ventilation-perfusion mismatch, or incomplete diffusion equilibrium (176). The rib cage and its muscles, the diaphragm, and the abdomen and its muscles make-up what is known as the ventilatory pump (160). Alveolar ventilation and gas exchange depend on the ventilatory pump. Hypercapnia is a telltale sign of ventilatory pump failure. A reduction in central neural drive, inspiratory muscle impairment, and/or excessive respiratory workload can induce ventilatory pump failure (3). Neuromuscular disease, drug overdose, or brainstem injury can impair central drive to the point of inducing ventilatory pump failure (3). Inspiratory muscle performance can be negatively impacted by neuromuscular disease (4), metabolic disturbances (3), certain drugs (2), a

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7 disadvantageous length-tension relationship (175), mechanical disadvantage (103), altered force-velocity relationship (3), detraining and atrophy (8, 92, 125), and fatigue (2, 135). Excessive inspiratory muscle workload due to obesity, asthma, or pulmonary resection for example, can also lead to respiratory pump failure. Increased inspiratory muscle workload results in an increased work of breathing. An increased work of breathing requires recruitment of the diaphragm (the primary muscle of inspiration) and also recruitment of the accessory inspiratory muscles. This presents two significant challenges: increased workload for the diaphragm and increased oxygen consumption. The diaphragm is well suited to the constant demand of pulmonary ventilation. However, if the constant workload exceeds 40% of its maximal force-generating ability, it will fatigue (135) and ventilatory pump failure may ensue. A further consideration of an increased work of breathing is the increased demand for oxygen by the respiratory muscles. The respiratory muscles may account for more than 50% of the total body oxygen consumption, as compared to less than 5% under normal conditions (13). The increased oxygen cost of breathing reduces the availability of oxygen to other body tissues and may lead to other potentially fatal events (e.g., myocardial ischemia) (167). As mentioned above, an increased work of breathing, if excessive, may lead to diaphragmatic fatigue and thus hypoventilation. Hypoventilation causes a loss of oxygen intake and also causes hypercapnia (which significantly impairs muscle contractility) (85). This series of events, if left unchecked, can put the patient into a downward spiral of respiratory muscle distress, hypoventilation, and hypercapnia (167). This scenario may be remedied, however, by placing the patient on MV and thus

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8 resting the diaphragm. In fact, mask ventilation typically improves respiratory frequency, arterial oxygen tension, and pH soon after it is applied (6, 31). Modes of Mechanical Ventilator Operation The mode of MV depends on the needs of the patient (central drive, respiratory muscle dysfunction, etc.). Controlled MV, assisted-control ventilation, intermittent mandatory ventilation, and pressure support ventilation are all modes of MV commonly used to aid the patient. The properties of each are outlined below. Controlled Mechanical Ventilation Controlled MV (CMV) is perhaps the most straightforward mode of MV. Controlled MV delivers all breaths in a predetermined fashion. Breathing frequency (f), tidal volume (V T ), inspiratory-to-expiratory timing (I:E ratio), and inspiratory flow pattern are each regulated by the ventilator settings. The patients breathing frequency or inspiratory effort cannot alter the preset respiratory parameters; hence, patient triggering is not possible during CMV. Controlled MV is achieved pharmacologically (e.g., sedation and neuromuscular blockade) or by mechanical hyperventilation (78). The use of CMV is limited to patients who are apneic because of brain damage, sedation, or neuromuscular blocking agents (167). Controlled MV is used to treat hypoxemic respiratory failure due to widespread atelectasis, localized alveolar disease, noncardiogenic pulmonary edema, and cardiogenic pulmonary edema (3). Controlled MV is also used to treat hypercapnic respiratory failure due to acute neuromuscular disease and acute obstructive disease (3). While CMV provides the maximum degree of respiratory muscle rest it is not without consequence to the very muscle it is intended to aid, the diaphragm. Administration of CMV often requires the use neuromuscular blocking agents whose use

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9 is associated with prolonged weakness or paralysis lasting up to 1 week (68, 89, 143). The effect of the neuromuscular blocking agents is compounded when corticosteroids are given. The combination of neuromuscular blocking agents and large doses of corticosteroids can result in generalized myopathy lasting weeks or months (44). Further, this pharmacologic combination is associated with a higher incidence of muscle weakness (93). Even in the absence of disease, CMV can induce muscle weakness which is attributed to muscle atrophy (167). Assist-Control Ventilation Assist-control (AC) ventilation is the first mode of MV used in many institutions (104). Assist-control ventilation provides a positive pressure breath in response to the patients inspiratory effort. The V T of each breath is set at the ventilator. The V T is delivered with each inspiratory effort or if the patient fails to trigger the ventilator within a set amount of time. Assist-control allows the patient to control breathing frequency. The patient also controls the ventilator-generated pressure. As the inspiratory effort generated by the patient increases, the ventilator-generated assistance decreases (104). Under the most favorable conditions, AC is 50-66% more effective than active chest inflation at reducing respiratory work of breathing by (108). Intermittent Mandatory Ventilation Intermittent mandatory ventilation (IMV) provides a preset number of positive pressure breaths and allows the patient to breathe spontaneously between ventilator-delivered breaths. Intermittent mandatory ventilation can be set to provide a breath that is either a preset volume or pressure. Once a predetermined pressure is reached, the ventilator terminates the positive pressure breath. Further, IMV allows the patient to autonomously alter his/her spontaneous breathing pattern. Because each IMV breath is

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10 synchronized with the patients breathing pattern, this mode of ventilation is also known as synchronized intermittent mandatory ventilation (SIMV). While IMV gives the clinician great flexibility in treating the patient, it has a potential drawback. Intermittent mandatory ventilation was designed to provide volume assistance while allowing the patient to breathe spontaneously (that is, to rest the inspiratory muscles and attenuate inspiratory muscle deconditioning). However, when IMV accounts for 20 to 50% of the total ventilation, the electromyographic (EMG) activity of the diaphragm and the sternomastoid muscles is equal to that of spontaneous breaths (81). While IMV is intended to provide inspiratory muscle rest, the EMG data suggest that this may not be the case. Pressure Support Ventilation Pressure support ventilation (PSV) is designed to augment the patients inspiratory effort by providing positive pressure support. Pressure support ventilation reduces the work of breathing by raising the airway pressure to a predetermined level after the patient initiates a breath; and continues to do so until the end of the inspiratory effort is sensed as a reduction in inspiratory flow (31). In PSV treatment, breathing frequency, V T and inspiratory flow pattern are determined by the patient. Pressure support ventilation is widely used in intensive care units because it does not require heavy sedation of the patient and it allows the patient to breathe spontaneously. Further, the patient is required to use their inspiratory muscles, thereby reducing the severity of inspiratory muscle deconditioning. Diaphragmatic Motion during Mechanical Ventilation The diaphragm, like the lung, can be described in terms of its position relative to the pressures acting on it. In the upright position there is a vertical gradient in pleural

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11 pressure acting on the lungs, and the pressure acting on the upper portion of the lungs is more subatmospheric than the pressure at the bases of the lungs. The region of the lungs at the bottom of the vertical gradient is termed dependent and the region at the top of the gradient is termed nondependent. Therefore, while in the upright position the bases of the lungs are in the dependent region, and the apices are in the nondependent region. Shifting the body position to the supine position changes the dependent and nondependent relationships (i.e., the dorsal region of the lungs is in the dependent region and the ventral surface of the lungs is in the nondependent region). Likewise, while in a supine position, the ventral portion of the costal diaphragm is in the nondependent position and the dorsal portion of the costal diaphragm and the crural portion of the diaphragm are in the dependent region. The middle costal portion of the diaphragm lies between both regions but is often considered to be dependent. While breathing spontaneously in the supine position, the dependent diaphragm is displaced or has a greater excursion than the nondependent diaphragm (54, 151, 87) because of anatomical differences between the costal and crural diaphragm region (87). After anesthesia and MV, the position of the diaphragm at functional reserve capacity (FRC) shifts cephalad (23, 54, 87, 129). The cephalad shift is due to loss of muscle tone in the diaphragm and gravitational displacement of the abdominal contents (23, 54, 129). After CMV the pattern of displacement is reversed: the dependent regions of the diaphragm are displaced less than the nondependent regions of the diaphragm (54). This reversal of displacement is the result of a uniform increase in thoracic pressure displacing the diaphragm where abdominal pressure is least (i.e., the nondependent

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12 region of the diaphragm) (54). Thus, during MV the diaphragm is passively moved each time a breath is artificially delivered to the patient. The diaphragm does not shorten to the same extent during MV as during spontaneous breathing. The diaphragm does, however, shorten passively while being displaced by the artificially ventilated lungs (119). The degree to which the diaphragm passively shortens during MV is not uniform. Diaphragmatic shortening during PSV (87, 129) and CMV (151) has been reported to be less than that of spontaneous breathing. However, others (53, 132) have reported greater diaphragmatic shortening during CMV than during spontaneous breathing (119). The disparity between these findings may be due to differences in methodology. The studies that reported less diaphragmatic shortening during MV used indirect methods such as 3-dimensional x-ray tomography (87), CT scans (129), and videofluoroscopy (151) to measure diaphragmatic length. The studies that reported greater diaphragmatic shortening during MV used Sonomicrometry (119). Sonomicrometry is a more accurate and perhaps more reliable method for measuring muscle length changes. Despite the different length changes reported during MV, the unifying finding is that the diaphragm shortens passively. In addition to passive shortening, the diaphragm is also displaced by the lungs during MV. Weaning from Mechanical Ventilation The common term for discontinuation of MV is weaning. This term refers to the slow withdrawal of MV at a rate the patient can tolerate. The weaning success rate in many intensive care units (ICU) is usually higher than 70% depending on the subset of patients (94). A person is considered a weaner if he/she is breathing spontaneously 2

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13 days after discontinuation of MV (94). A patient who requires some degree of ventilatory support (total or partial) is considered a non-weaner (94). It is imperative to discontinue use of MV as soon as possible because MV is associated with several major complications. Most patients requiring short-term MV experience little difficulty when MV is withdrawn. As previously discussed, MV is frequently used to aid patients recovering from respiratory failure. Discontinuing MV for many of these patients is difficult. Because of the factors that led to the patients placement on MV, great care is given to their discontinuation from MV. The process of discontinuation from MV is challenging and makes up a large portion of the ICU workload (166). The process of weaning may require more than 2 days and it can become a lengthy process. A long-term ventilator-assisted individual is a person who requires mechanical ventilatory assistance for more than 6 hours a day for more than 3 weeks after all acute illnesses have been maximally treated and in whom multiple weaning attempts by an experienced respiratory care team have been made (105). While MV may not be the primary reason a patient is in the hospital, it is often the reason for a prolonged stay. The total number of difficult weaners ranges from 20% up to 70% in some ICUs (94) and the cost to treat these patients is large (105). A 1983 study estimated that there were 6,800 long-term MV patients at a cost of ~$1.7 billion per year or ~1.5% of total hospital costs (106). In 1990, the American Association for Respiratory Care commissioned a rigorous study of the incidence and cost of long-term MV care (7). This study found that there were 11,419 long-term MV patients at an annual cost of $3.2 billion for treatment (7).

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14 While these studies relay the magnitude and dollar cost of MV, the importance of the loss of individual independence as well as the emotional cost should not be forgotten. Properties of the Diaphragm The diaphragm is the primary muscle of inspiration. As such, it is chronically active and its metabolic characteristics reflect this. This section reviews the functional and metabolic characteristics of the diaphragm. Function of the Diaphragm Breathing has long been recognized as a vital process. For example, around 2000 BC, Chinese philosophers wrote about lien chi, the process of bringing the inspired breath into the soul substance (35). The ancient Greeks also believed that breathing was essential and that the diaphragm was the seat of the soul (102). The Greek word phrenes means soul; this is how the phrenic nerves were named (102). In the third century BC, the diaphragm was recognized to be a muscle by Erasistratus and he taught that it was the main muscle of inspiration (43). The diaphragm is chronically active skeletal muscle and is innervated by the phrenic nerves from the cervical segments 3, 4, and 5 (176). The diaphragm has two functionally different and distinct parts: the central tendon (the non contractile portion) and the costal and crural regions (the muscular portions). The costal and crural muscle fibers extend outward from the central tendon. The fibers of the crural diaphragm radiate from the central tendon and insert onto the anterolateral aspect of the first three lumbar vertebrae and the aponeurotic arcuate ligaments (39). The fibers of the costal diaphragm extend from the central tendon and insert on the xyphoid process of the sternum (the ventral region) and the upper margins of the lower 6 ribs (the medial and dorsal regions)

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15 (39). The muscle fibers of the costal diaphragm run cranially from their insertions and are thus apposed directly to the inner aspect of the lower rib cage (39). Chest wall displacement during inspiration is accomplished by the unique shape and location of the diaphragm. The healthy diaphragm is an elliptical cylinder capped by a dome (39). The dome region is primarily composed of the central tendon. The cylindrical region is the portion apposed directly to the inner aspect of the lower rib cage, the zone of apposition (39). This shape and location gives the diaphragm the ability to increase chest wall dimensions and therefore inflate the lungs (123). Activation of the diaphragm elicits a caudal force onto the central tendon and a cephalic force onto the lower 6 ribs by the costal diaphragm and vertebral column by the crural diaphragm (123). The caudal force causes the dome of the diaphragm to descend and displace the abdominal contents downward, but the abdominal contents resist displacement and therefore act as a fulcrum (160). Thus, as the diaphragm contracts and its fibers shorten, the transverse dimensions of the chest wall increase (123, 160). Contraction of the diaphragm also increases the cephalo-caudal dimensions of the chest wall (123). Based on the insertions of the costal and crural diaphragm, the chest wall dimensions are changed by the costal region as the crural region inserts onto the immovable vertebral column and only displaces the abdomen (123, 160). Metabolic Characteristics of the Diaphragm The anatomical and morphological design of the diaphragm is well suited to the constant demand of the ventilatory system. Likewise, the metabolic design of the diaphragm allows it to meet the constant challenge imposed on it.

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16 Skeletal Muscle Fiber Types within the Diaphragm Skeletal muscle is a highly plastic tissue (it has the ability to adapt to the workload imposed on it). Skeletal muscle meets the workload by expressing fiber types best suited to the demand. Thus, the heterogeneity of skeletal muscle fibers expressed within a muscle is a reflection of the job the muscle is responsible for. The diaphragm is a highly specialized skeletal muscle. It is the only skeletal muscle that is chronically active for life and its fiber type expression reflects this. Fiber typing has evolved a great deal beyond the initial classification of red and white put forth by Ranvier in 1873 (128). As reviewed by Pette and Staron (120) muscle fibers can be classified by various methods including histochemistry, immunohistochemistry, and gel electrophoresis. Histochemical classification is a subjective method based on myofibrillar actomyosin adenosine triphosphatase (ATPase) activity or aerobic and anaerobic metabolic enzymes. These methods typically reveal 3 fiber types: I, IIa, and IIb (via ATPase) or slow-twitch oxidative, fast-twitch oxidative glycolytic, and fast-twitch glycolytic (via enzymatic analysis) (120). Immunohistochemistry and gel electrophoresis are able to resolve 4 fiber types based on myosin heavy chain (MHC) proteins: I, IIa, IId/x, and IIb. Immunohistochemistry and gel electrophoresis have been used to determine the fiber type composition of rat and human diaphragm (). Table 2-1 Overall however, the human and the rat diaphragm are very similar in fiber type composition (49). Recently, the MHC content of human single muscle fibers were thoroughly characterized histologically, immunohistologically, and electrophoretically (46, 140). This information was then correlated with the mRNA transcripts for each MHC gene.

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17 Table 2-1. Fiber type composition (%) of the diaphragm and locomotor skeletal muscles. T yp e Muscle I IIa IId/x IIb Method Reference Rat DIA 30 50 20 HC 49 COD 42 27 32 HC 111 COD 32 28 40 HC 63 COD 44 8 18 32 HC 41 COD 42 32 26 HC 124 DIA 14 32 53 1 SDS-PAGE 19 COD 28 31 38 3 SDS-PAGE 156 COD 26 21 41 12 SDS-PAGE 124 COD 35 30 30 5 IH 90 COD 24 23 43 10 IH 145 PL 9 50 41 HC 11 PL 3 0 46 51 SDS-PAGE 19 PL 3 14 40 43 SDS-PAGE 157 PL 4 18 50 28 SDS-PAGE 124 SOL 87 13 0 HC 11 SOL 94 6 0 0 SDS-PAGE 157 SOL 100 0 0 0 SDS-PAGE 19 Human DIA 55 20 25 HC 49 COD 55 21 24 HC 100 COD 49 28 23 HC 114 COD 43 41 17 SDS-PAGE 163 COD 45 39 17 SDS-PAGE 97 COD 46 39 18 IH 97 SOL 78 22 0 HC 69 SOL 70 30 0 SDS-PAGE 69 VL 47 41 12 HC 69 VL 47 49 4 SDS-PAGE 69 All values reported are percentages. Because of rounding, values may not total 100%. DIA = whole diaphragm. COD = costal diaphragm. PL, plantaris; SOL, soleus; VL, vastus lateralis; HC, histochemistry; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IH, immunohistochemistry. The mRNA transcripts in all fibers classified as IIb using conventional methods are actually equivalent to the rat IIx gene (46, 140). Based on these findings it was suggested that all type II fibers previously identified as IIb would be more accurately classified as

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18 IIx (46, 140). Additionally, conventional histochemical techniques may have erroneously classified rat IIx fibers as IIb (120). In light of these findings, the diaphragms from the rat and human are quite similar, as neither has many type IIb fibers. Oxidative Capacity The oxidative capacity of skeletal muscle is consistent with its function. Skeletal muscles that contract tonically are slow twitch and highly oxidative, whereas muscles used sporadically are fast twitch and have a low oxidative capacity. The characteristics of the diaphragm fit this biochemical tenet. Most muscle fibers in the diaphragm are highly oxidative and this is in-line with the predominant fiber types in the diaphragm, type I and IIa. Typical biochemical markers of skeletal muscle metabolic pathways include citrate synthase (CS), succinate dehydrogenase (SDH), 3-hydroxyacyl-CoA dehydrogenase (HADH), hexokinase (HK), phosphofructokinase (PFK), and lactate dehydrogenase (LDH). The oxidative capacity of a muscle is often estimated by the citric acid cycle enzymes CS and SDH. Likewise, the lipolytic capacity can be determined by an integral -oxidation enzyme, HADH. The enzymes HK, PFK and LDH are often used to determine the glycolytic capacity of muscle cells To date, there are two published reports of normal human diaphragmatic bioenergetic enzyme capacities (139, 163). Sanchez et al. (139) compared the bioenergetic capacity of the diaphragm to the latissimus dorsi. In each case, thebioenergetic capacity of the diaphragm was significantly greater than that of the latissimus dorsi (i.e., CS 180%, HADH 215%, HK 170%, and LDH 115%).

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19 Table 2-2 shows the bioenergetic similarities of the human and rat costal diaphragm, as well as two different locomotor muscles. Note that the bioenergetic capacity of the human and rat diaphragm are very similar. Overall, the bioenergetic capacity of the diaphragm reflects its continuous use, a high oxidative capacity as measured by citric acid cycle and -oxidation enzymes, and a moderate glycolytic enzyme capacity. Table 2-2. Bioenergetic enzyme activities in the costal diaphragm and two locomotor muscles Muscle CS HADH LDH Reference Human COD 0.33 0.27 11.6 (163) Rat COD (Rat) 0.46 0.23 4.8 (126) PLA (Rat) 0.29 0.11 8.4 (126) SOL (Rat) 0.33 0.22 2.5 (126) COD, costal diaphragm; PL, plantaris; SOL, soleus; CS, citrate synthase; HADH, 3-hydroxyacyl-CoA dehydrogenase; LDH, lactate dehydrogenase. Enzyme activities are expressed as M/min/mg of protein. Muscle Atrophy The diaphragm, like all skeletal muscle, is highly plastic and therefore rapidly adapts to the demands placed on it. Skeletal muscle quickly adapts to alterations in proprioceptive activity, motor innervation, mechanical load, and joint mobility (10). Skeletal muscle adapts to an increase in muscular activity by increasing contractile and structural protein content (hypertrophy), whereas inactivity or disuse leads to a loss of muscle mass (atrophy) (10). Hypertrophy (protein accumulation) and atrophy (net loss of protein), therefore, are critically dependent on the relative rates of protein synthesis and protein degradation (61). Atrophy results in a decrease in cross sectional area (CSA) and this is functionally significant because muscle strength is directly related to CSA (26). MV is a common method of reducing or removing diaphragmatic work. The reduction in workload by MV leads to diaphragmatic disuse muscle atrophy and/or

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20 weakness and is a major mechanism of weaning failure (167). The term disuse is relative and can be defined as a reduced level of contractile activity (116). Two characteristic components of reduced contractile activity include hypokinesia and hypodynamia (116). Hypokinesia refers to a decreased level of contractile activity (i.e., reduced limb movements) and hypodynamia is a decrease in mechanical loading (i.e., reduced weight-bearing function) (116). MV and other models of skeletal muscle disuse atrophy are reviewed below. Models of Locomotor Muscle Atrophy Several different experimental models and human clinical conditions result in skeletal muscle atrophy. These models and conditions will be reviewed according to the level of neuromuscular activation altered by electrical activation and weight-bearing status. Reduced Electrical Activation and Load Bearing The electrical activation and load bearing status of skeletal muscle can be altered by spinal cord injury, spinal cord transection, and limb immobilization (with the muscle of interest in the shortened position). In this section, spinal cord injury (SCI) and spinal cord transection (ST) will be considered together. Spinal cord transection interrupts the upper motor neuron pathway by transecting the spinal cord, often at the thoracic level. In cat soleus, ST results in a 75% reduction in the daily-integrated electromyographic (EMG) activity and a 66% reduction in the total duration of the EMG (1). After ST, the ankle joint of the animal is held in an extended position, effectively unloading the soleus (1). After 5 and 10 days of ST, the rat soleus significantly atrophies, ~35% and 60% reduction in the total CSA respectively (45). Further, ST alters myosin expression from slow-to-fast MHC at both the mRNA (45) and protein level in the soleus (45, 138). Six to

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21 8 months after ST biochemical measures such as citrate synthase and myosin ATPase activities become characteristic of fast muscle (138). The functional outcome of ST is a loss of absolute force generating ability by the affected muscle(s). The absolute tension generation by the medial gastrocnemius and soleus after 6 to 8 months of ST is reduced by 26% (137) and 50% respectively (138). Human skeletal muscle also undergoes a slow-to-fast MHC shift After SCI. The vastus lateralis from 15 patients was sampled 6, 11, and 24 weeks after SCI (33). At the 6 and 11 week time point there was no detectable MHC shift but after 24 weeks there was a 16% increase in type IIx MHC expression (33). Thus, human skeletal muscle adapts to reduced electrical activation and muscle loading but at a slower rate than small animals such as rat and cat. Overall, ST and SCI lead to alterations in fiber type composition and impairs force generation. Hindlimb immobilization (HI) is a clinically relevant model of reduced electrical activity and loading. Hindlimb immobilization is achieved by fixing the joint(s) at a specific angle by either pinning the joint(s) surgically with steel rods or by fixing the limb with orthopedic plaster. The electrical activation of the rat soleus after HI in the neutral position is ~50% of control (52). Similarly, the electrical activity of the human quadriceps is ~60% less than the electrical activity observed in control quadriceps (77). Atrophy induced by HI results in significant decrements in the CSA of both rat (9) and human (77) skeletal muscle within 7 to 21 days, respectively. Accordingly, the decrease in CSA results in an ~50% loss of strength in rat soleus (177) and human quadriceps (77) skeletal muscle. Metabolic markers such as citrate synthase and lactate dehydrogenase indicate that HI shifts the normally slow oxidative soleus toward a fast oxidative

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22 glycolytic muscle (51). Further, the slow-to-fast MHC shift occurs rapidly at the mRNA level. After 1 week of HI soleus IIx and IIb mRNA transcript levels increased ~24 and 2.6 fold compared to control (83). Fiber type conversion in human skeletal muscle After HI also follows the slow-to-fast pattern. After 3 weeks of HI, type I mRNA transcripts were ~30% less than control and IIx transcripts were ~300 times greater than control (77). To date, there are no data on the levels of MHC protein isoforms after HI in either the rat or humans. Within hours of HI, protein synthesis and protein degradation are altered. Six hours after HI the fractional rate of protein synthesis in the soleus is reduced 20-35% (28, 60). For example, Goldspink (60) measured a 20% decrease in the synthetic rate with ~2% loss of muscle wet weight and a slight decrease in the rate of protein breakdown after 6 hours of HI. After 2 days of HI, soleus wet weight was reduced by 25%, the rate of protein synthesis was 65% less, and the rate of protein degradation was 50% greater than control (60). Booth (28) reported similar findings after 6 hours of bilateral HI, a ~35% decrement in the fractional rate of protein synthesis in the gastrocnemius. Reduced Loading Due to the limited nature of spaceflight, ground based models such as hindlimb unloading (HU) of the rat, and human bed-rest models have been devised as models of reduced loading. Hindlimb unloading of the rat is typically achieved by placing a plaster cast at the base of the tail to which a gimbal is attached. The animal is then raised such that the forelimbs support the weight of the animal and its hindlimbs are not in contact with the ground. To date, EMG recordings of rat hindlimb activity have not been made during actual spaceflight. However, EMG recordings of rat soleus during parabolic flight, ~25

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23 sec of micro-gravity, is ~10% of control values (95). This rapid decrease in EMG activity has also been observed using HU. The EMG activity of the soleus is nearly abolished ~3 sec after HU is imposed (5). Chronic soleus EMG activity either remains decreased (24) or returns to normal (5) after 28 days of HU. While actual force measurements have not been made during spaceflight or HU, it is generally accepted that force generation is minimal (159). The contractions are spontaneous isotonic contractions and do not generate much force because they are not weight bearing and are similar to the contractile patterns that occur in the muscles of astronauts in space (27). Spaceflight and HU rapidly affect muscle weight. After 5 to 7 days of spaceflight, rat soleus mass is ~18 to 30% less than control (75). Additionally, soleus muscle fiber CSA is significantly reduced (-14%) and absolute tension is significantly impaired (-28%) (75). Hindlimb unloading induces similar outcomes after 4 to 5 days, ~15-30% loss of soleus mass (101, 155). Seven days of HU significantly reduces type I fiber CSA, -14%, and absolute tension, -30% (110). Lactate dehydrogenase activity is not altered by spaceflight, but HU significantly decreases its activity, -27% (117). Conversely, citrate synthase activity is not affected by either spaceflight or HU (117). While the unloading effect significantly alters slow antigravity muscles such as the soleus, the morphometry and function of the fast extensor digitorum longus is not impacted by 14 days of spaceflight (154). The change in MHC phenotype in the HU soleus is a very predictable slow-to-fast shift. After 4 days the loss of type I MHC is small, but after 7 days the loss is significant (155). The increase in type IIa MHC expression is significant after only 4 days (155). After 7 days, type IId/x MHC is significantly increased and an increase in type IIb is

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24 found after 14 days (155). The expression of each MHC mRNA transcript roughly mirrors protein expression. A small change in type I MHC mRNA (the mRNA transcript for type I MHC) expression occurs after 4 to 7 days and a significant increase in the expression of I mRNA (the transition mRNA transcript from I to IIa) occurs in the same time period (155). Type IIa mRNA expression increases slightly and IId/x and IIb expression significantly increases within 4 days (155). The decrease in type I and IIa mRNA (>30%) and the increase in IId/x and IIb (>100%) mRNA becomes significant after longer periods of time, 9-14 days in space (32, 67) and thus, the slow-to-fast MHC shift rapidly occurs at both the protein and mRNA level. A contributory factor leading to the loss of muscle mass under reduced loading conditions is the decreased rate of protein synthesis. Total mixed protein synthesis and myofibrillar protein synthesis measured during the first 5 hours of HU decreases 16% and 22% respectively in the rat soleus (162). In these experiments protein synthesis was measured by constant infusion of radio-labeled leucine into the animal over the 5 hour period. The measurement of protein synthesis during the first 5 hours of HU was therefore an average of the initial response and may not reflect the true rate during the 5 th hour. However, after 24 hours of HU, total and myofibrillar protein synthesis in the soleus is significantly diminished, -30 and -15% respectively (115). Thus, skeletal muscle adapts rapidly to HU by decreasing protein synthesis and importantly, the rate of myofibrillar protein synthesis. Models of Inactivity Inactivity includes spinal cord isolation and blockage of the motoneuron action potential conduction by substances such as tetrodotoxin. Also,denervation is often

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25 included as a model of inactivity. Denervation is achieved by severing the motoneuron-muscle connection, but because it also interrupts neural input to tissues such as vascular tissue, thus altering blood flow, its effect is often difficult to interpret (116) and will not be considered here. Spinal cord isolation (SI) has been used to study the effects of nueromuscular activity on skeletal muscle. Spinal cord isolation involves complete spinal cord transection at the mid or low thoracic and lumbar-sacral levels, as well as complete deafferentiation between the two points of transection (159). This preparation maintains the integrity of the neuromuscular unit and yet eliminates sensory input from the dorsal roots as well as neural signals from either above or below the transection (159). The atrophic effect of SI is severe and rapid. The soleus loses 25% of its mass and 75% of its fiber CSA 4 days after SI (64). The slow-to-fast shift in MHC is relatively slow, resulting in a 10% increase in IId/x expression after 15 days and a 40% loss of type I MHC after 60 days (64). Spinal cord isolation causes skeletal muscle to become not only smaller and faster, but weaker as well. Maximum force generation by the soleus 6 months after SI is decreased by 80% (136). The enzymatic profile of skeletal muscle shifts from oxidative (-70% succinate dehydrogenase activity) toward glycolytic (+120% glycerolphosphate dehydrogenase activity) after SI (84). To date, there are no published reports of total and/or myofibrillar protein synthesis After SI, however, the rate and severity of SIinduced atrophy suggests that both would be decreased. Tetrodotoxin (TTX) is a potent Na + channel blocker and if applied continually to motor nerves it will inactivate skeletal muscle. Muscle paralysis after TTX treatment, as determined by EMG activity during locomotion, is nearly complete in all treated animals

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26 (152). This model of inactivity leads to significant atrophy, 46.5% decrease in muscle mass and a 25% decrease in myofibrillar protein in 2 weeks (152). Concomitant with the TTX-induced atrophy is a 38% loss of force generating ability (152). The expression of MHC is altered by TTX treatment such that the expression of type I MHC increases, type IIa decreases, type IId/x increases, and type IIb does not change (112). It is currently unknown why type I MHC expression increases in response to TTX treatment but it may be related to the disruption in the delivery of a neurotrophic factor to the motor end-plate (159). Tetrodotoxin treatment severely diminishes the metabolic capacity of both oxidative (citrate synthase %) and glycolytic (phosphfructokinase % and -glycerolphosphate dehydrogenase %) enzymes (153). The effect of TTX treatment on protein synthesis is currently unknown but it likely plays a role in the observed atrophy response. Models of Atrophy in the Diaphragm The primary muscle of inspiration is the diaphragm and it is chronically active throughout life. Due to its chronic activity the activation pattern of the diaphragm differs from locomotor muscles such as the extensor digitorum longus (EDL) and the soleus. The rat diaphragm has a duty cycle (duration of inspiratory time/total respiratory cycle duration) of ~40% (141) while the EDL and soleus have duty cycles of 2% and 14% respectively (71). Thus, the contractile history of the diaphragm differs greatly from locomotor muscles and it may be more susceptible to alterations caused by inactivity (113). Models of diaphragmatic inactivity include denervation, blockade of nerve impulses by TTX, spinal cord transection (ST), and MV. The effects of MV will be considered separately from the other models.

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27 Procedure for Investigating Diaphragmatic Atrophy Diaphragmatic denervation (DNV) is accomplished by dissecting the phrenic nerve, unilaterally or bilaterally. Once dissected, the phrenic nerve is transected (phrenicectomy) and a significant portion is removed (e.g. ~10-20 mm in the rat) to prevent possible reinnervation of the diaphragm (98). Again, TTX is a Na + channel blocker that prevents action potential propagation. The phrenic nerve is typically dissected at the lower neck and a Silastic cuff is placed around the nerve and connected to a miniosmotic pump that continuously perfuses the nerve with TTX. Spinal cord transection (ST) is achieved by performing a dorsal laminectomy after which one-half (e.g. right side) of the cervical spinal cord (e.g. at C 2 ) is sectioned from the dorsal root to the ventral root. Correctly done, only the ventral and lateral funiculi are cut and the lateral funiculus is preserved in order to minimize motor deficits in the ipsilateral side (113). Electromyographic Activity Paralysis of the diaphragm is verified by the EMG activity of the left and right sides (hemidiaphragm) of the diaphragm. The EMG signal is obtained by implanting small diameter wire electrodes into the diaphragm. Each of the above-mentioned surgical preparations induces diaphragmatic paralysis (i.e., no EMG activity) of the intended hemidiaphragm, and a 50% increase in the activity of the intact hemidiaphragm due to a compensatory increase in muscle activation (183). However, phrenic nerve activity after unilateral DNV and unilateral TTX significantly increases ~40-50% on both sides (113). The increase in activity indicates that there is a compensatory increase in central drive to motor neurons on both sides of the spinal cord (113). The ST model, however, leads to

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28 inactivity of the phrenic motoneurons (113). Diaphragm paralysis and motoneuron activity are matched in the ST model, whereas motoneuron activity and paralysis are not matched in the TTX model, and the connection between the nerve and muscle is obviously severed in the DNV model (113). The morphological, biochemical, and mechanical alterations that result from each of the above models is attenuated when the activity of the diaphragm muscle fibers is matched by the phrenic motoneuron. Diaphragmatic Mass The initial response to diaphragmatic paralysis via DNV is hypertrophic after which the response becomes atrophic. After 8 days of unilateral and bilateral DNV, the diaphragm hypertrophies 20% (paralyzed hemidiaphragm) and 13% (each hemidiaphragm hypertrophied) respectively (179). The hypertrophic response is diminished by the second week and diaphragmatic mass returns normal (185). The chronic response (i.e., 6 weeks) to diaphragmatic paralysis is a significant loss of diaphragmatic mass (-37%) (98). To date, the effect of ST and TTX on diaphragmatic mass has not been determined. Differential Fiber Type Response to Diaphragmatic Inactivity The change in cross-sectional area (CSA) after unilateral hemidiaphragm paralysis induced by ST, TTX, and DNV is variable; in each case the alterations brought about by ST are not as dramatic as those of TTX and DNV after 2 weeks of treatment. Type I fiber area hypertorphies 33, 70 and 80% according to treatment, ST, TTX, and DNV, respectively (184). Type IIa fibers also hypertrophy 13, 99, and 81% after ST, TTX, and DNV respectively, (184). Paralysis induced by TTX and DNV leads to the appearance of hybrid type I/IIa fibers (184). Type IId/x fiber CSA is reduced 5, 34, and 39% and type IIb fibers also atrophy 15, 53, and 57% after 2 weeks of ST, TTX, and DNV respectively

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29 (184). The variable response to the treatments may be the result of better matching between muscle fiber and motoneuron activity in the ST model. After 6 weeks of unilateral DNV, the CSA of the type I and IIa fibers returns to normal, whereas the IIb/x fibers lose ~57% of their CSA (98). The hybrid I/IIa fibers that appear after 2 weeks of DNV (184) occupy ~50% of the total CSA after 6 weeks of DNV (98), indicating a slow-to-fast shift in MHC. Myosin Heavy Chain Content Diaphragmatic MHC content has been followed over a 3-week period of DNV (147). Type I MHC increases after 1 week (+30%) and begins to decrease during the 2 nd (+25%) and 3 rd week (-6%) (147). The type IIa MHC expression is elevated during the first 2 weeks, +23% week one and +29% week two, and begins to return to normal by the 3rd week, +11% (147). The expression of type IId/x MHC falls off rapidly during the first 2 weeks, -27% and %, and then begins to return to normal by week three, -6% (147). Type IIb MHC expression declines rapidly and remains depressed over the entire 3-week period, -50% to -66% (147). Myosin Heavy Chain mRNA Northern analysis of MHC mRNA reveals a down regulation of all 4 MHC transcripts after 8 days of DNV (179). Type I, IIa, IId/x, and IIb mRNA levels significantly decrease 50, 70, 60, and 35% respectively after 8 days of reduced activity (179). This is an interesting observation when one considers the finding that type I and IIa MHC content increases and IId/x and IIb expression decreases at the protein level (147). These observations suggest differential posttranscriptional regulation of the four MHCs.

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30 Contractile Properties After 2 weeks of unilateral hemidiaphragm paralysis induced by ST, TTX and DNV, specific tension declines 23, 49, and 51% respectively, (113). In accordance with the observed, type I and IIa hypertrophy (184) and the increase in type I and IIa MHC expression after diaphragmatic paralysis, diaphragmatic contraction and relaxation times dramatically increase (113). Additionally, actomyosin ATPase activity is reduced by all three treatments (184). Again, the effects of ST were not as pronounced as those induced by TTX or DNV (113, 184). Oxidative Capacity Succinate dehydrogenase (SDH), a marker of oxidative capacity, is significantly reduced after 2 weeks of ST, TTX and DNV (184). The reduction in SDH activity did not occur in all fiber types however. The oxidative capacity was not altered in the type I or IIa fibers. Paralysis induced by TTX and DNV reduced SDH activity in type IId/x and IIb fibers whereas ST only reduced SDH activity in the IId/x fibers (184). Protein Synthesis To date, measures of protein synthesis have only been performed using the DNV model of disuse. As might be expected, in vivo protein synthesis is significantly increased (~50%) during the initial hypertrophic phase, days 1 through 10 (169). The significant increase in protein synthesis after acute DNV has also been observed under in vitro conditions (170). However, after approximately 3 weeks of DNV, the diaphragm atrophies significantly (29). While the rate of protein synthesis has not been measured after chronic diaphragm DNV, measures of diaphragmatic total RNA content have been made. During the hypertrophic period as well as the atrophic period, followed out for 51 days, total RNA content in the diaphragm parallels diaphragmatic mass (29). The

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31 decrease in total RNA content and the loss of diaphragmatic mass suggests that protein synthesis would be suppressed. Diaphragm Length Changes It has been hypothesized that passive stretch is an underlying mechanism responsible for the morphological adaptations incurred by the paralyzed side of the diaphragm (183). The paralyzed hemidiaphragm is pulled along by the intact hemidiaphragm as it contracts. Due to differences in muscle fiber orientation, the length changes that occur after unilateral paralysis varies between regions of the diaphragm. The muscle fibers in the paralyzed midcostal region of the diaphragm are stretched ~3-5% beyond resting length (diaphragm muscle fiber length at end expiration) because they are in series with the muscle fibers in the intact hemidiaphragm. The sternal region is passively shortened 4-5% of resting length because the muscle fibers are in parallel with the fibers of the opposite side (183). However, the degree of adaptation, as measured by in vitro contractile properties and fiber typing, was similar between regions, costal and sternal. If alteration of muscle fiber length, stretching or shortening, was the underlying cause for the observed changes in in vitro contractile properties after DNV, a differential response (e.g., passive shortening would induce greater contractile dysfunction) would be predicted. This was not the case and the authors suggest that removal of innervation itself is the underlying mechanism leading to the morphological adaptations (183). This observation is supported by earlier findings that reported less pronounced morphological and contractile adaptations after ST as compared to TTX or DNV (184). This suggests that interactions between the motoneuron and muscle fibers may play an integral role in muscle adaptation (183).

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32 Mechanical Ventilation and Diaphragmatic Atrophy MV is used to either fully support or augment alveolar ventilation. Further, MV rests the diaphragm and in doing so the phasic activity of the diaphragm is decreased by varying degrees, depending on the level of mechanical support. Electrical Activity Inhibition of diaphragmatic EMG activity during controlled MV (CMV) has been demonstrated in healthy subjects (91, 107, 148, 149) and in chronic obstructive pulmonary disease (COPD) patients (30). Likewise, MV reduces neuronal activity in regions of the brain that are known to be involved in the control of breathing (48). Importantly, the loss or reduction of EMG activity of a muscle is a primary factor in the etiology of disuse atrophy (e.g., 45, 52). Diaphragmatic Disuse Atrophy Mechanical ventilation is frequently used in caring for neonates and infants. The CSA of muscle fibers from the diaphragms of neonates and infants ventilated for more than 12 days before death is markedly smaller, -70% in one case, and consistent with disuse atrophy (86). Diaphragmatic atrophy may also be observed after cervical fracture. Patients with lesions above the origins of the phrenic nerve roots require ventilatory assistance (e.g., MV or phrenic nerve pacing). Measures of diaphragm thickness have been performed on patients treated with phrenic nerve pacing. The normal adult diaphragm is ~0.3 cm thick (109) and after 8 months of MV the thickness can decrease to ~0.18 cm (14). After 6 weeks of phrenic nerve pacing, diaphragmatic thickness can increase nearly two-fold with a three-fold improvement in maximal tidal volume (14). While this is not conclusive evidence of diaphragmatic atrophy induced by prolonged MV, it is highly suggestive of diaphragmatic atrophy.

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33 Animal Models and Mechanical Ventilation Due to the invasiveness of intubation and diaphragmatic biopsies, animal models have been devised to study the mechanical and biochemical effects of MV on the diaphragm. Anzueto et al. (8) tested the hypothesis that prolonged MV would impair diaphragmatic contractile properties. The investigators ventilated healthy adult baboons for 11 days and measured maximal transdiaphragmatic pressure (Pdi max ), the force frequency response, and endurance time preand post-MV (8). The ability of the diaphragm to generate force in vivo can be determined by measuring Pdi max This measurement of diaphragmatic strength was impaired by 11 days of MV, -32% to -48%. The diaphragmatic response to phrenic nerve stimulation over a wide range of frequencies (i.e., the force frequency response) was dramatically shifted downward after 11 days of MV (8), indicating a loss of force generating ability across a spectrum of stimulation frequencies. Fatigue resistance was assessed by requiring the animals to breath through a resistor at 60-70% of Pdi max until the target pressure could no longer be met. Diaphragmatic endurance time was reduced by ~36% after 11 days of MV (8). It is possible that the use of a neuromuscular blocking agent during the MV period contributed to the deleterious effects of MV. The investigators did however control for this by withholding the neuromuscular blocking agent for 8 hours before contractile measurements were made. Contractile measurements were conducted only after spontaneous breathing resumed and the diaphragm responded to phrenic nerve stimulation (8). Typically, muscle weakness occurs when neuromuscular blocking agents are used in conjunction with corticosteroids (36, 93) and only rarely occurs in patients not treated concurrently with corticosteroids (59). Nonetheless, the decrease in diaphragmatic

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34 force generation is similar to the results of Le Bourdelles et al. (92) and Powers et al. (125) who did not use neuromuscular blocking agents to study the effects of MV. The rat has also been used to investigate the impact of MV on the diaphragm. After 48 hours of MV, the mechanical properties of the diaphragm, soleus, and EDL as well as their biochemical properties have been documented (92). MV did not significantly alter body mass but the mass of the diaphragm, soleus, and EDL were significantly decreased after 48 hours of MV (92). MV did not affect the contractile properties of the soleus or EDL (92). However, diaphragmatic contractility was significantly impaired by 48 hours of MV. MV resulted in a downward shift in the force frequency response and reduced maximal tetanic tension ~60% (92). Further, MV negatively impacts indicators of calcium handling within the muscle such as the rate of force development (dP/dt) and the rate of relaxation (92). The slowing of these indicators suggests a decreased rate of calcium release and sequestration. MV did not affect the bioenergetic enzymes, citrate synthase and lactate dehydrogenase in the diaphragm (92). This indicates that the observed diaphragmatic dysfunction is not necessarily due to a derangement of metabolism but rather, an alteration in one or more steps of excitation-contraction coupling. Effects of Mechanical Ventilation on Diaphragmatic Contractile Properties Recently, our laboratory characterized the time course of MV induced diaphragmatic dysfunction (125). Animals were mechanically ventilated in the control mode for 12 to 24 hours. At the specified time point, a segment of the costal portion of the diaphragm was carefully dissected and used for in vitro contractile measurements. The body mass of each animal was measured preand post-MV and there was no change (p > 0.05). Additionally, the mass of the soleus did not differ between control and MV

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35 animals (p > 0.05). Arterial blood pressure, blood gases, and pH status were monitored throughout the MV period and were found to fluctuate, but remained within a narrow physiological range. Maintenance of blood gas and pH homeostasis is critical because hypoxia, hypercapnia, and respiratory acidosis are known to impair diaphragmatic function. Our findings reveal that contractile dysfunction occurs in as few 12 hours. Twitch tension was 35% less than control after 12 hours of MV. Twitch tension continued to fall over the 24-hour period; at which point, the twitch tension of the mechanically ventilated diaphragms was 42% less than control. The force frequency response of the mechanically ventilated diaphragms was impaired, shifted to the right, at all stimulation frequencies. The magnitude of the right-shift was exacerbated with time on the ventilator. MV significantly reduced (p < 0.05) maximal specific tension and the loss of force generating ability fell over time (i.e., % after 12 hours and % after 24 hours of MV). Our results of a 46% loss of specific tension are comparable to those of Le Bourdelles et al. (92) who reported a 60% loss of specific tension after 48 hours of MV. Further, our results indicate that the effect of MV is confined to the diaphragm as there was no loss of soleus muscle mass. This observation is similar to the finding of Le Bourdelles et al. (92) who reported no change in soleus or EDL maximal specific tension after 48 hours of MV. Mechanical Ventilation and Diaphragmatic Atrophy After the time course experiments, we tested the hypotheses that short-term MV (18 hours) would induce atrophy in all four fiber types in the diaphragm, increase the rate of diaphragmatic muscle protein degradation, and increase oxidative stress in the diaphragm (145).

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36 After 18 hours of MV there was no loss of body or soleus mass. However, total diaphragmatic mass was significantly reduced (-6.9%) and this was primarily due to the significant loss of costal diaphragmatic mass (-7.3%) (145). The atrophic effect of 18 hours of MV was confirmed using immunohistological techniques: the CSA of the type I fibers was reduced -15%, IIa -27%, IId/x -30%, and IIb -24% (145). Again, these findings support the postulate that MV results in diaphragmatic atrophy. Measures of total and myofibrillar protein content were made to determine if the observed contractile dysfunction and decreased diaphragmatic mass could be explained by alterations in the protein composition of the diaphragm after MV. Eighteen hours of MV resulted in significant reductions in diaphragmatic protein. Specifically, the concentration of both myofibrillar protein and soluble protein significantly decreased by ~10%, resulting in a significant decrease in the total protein concentration (145). Consistent with the loss of diaphragmatic mass, was the reduction in total (-16%) and myofibrillar protein content (-16%), reflecting an absolute loss of protein from the diaphragm (145). Additionally, MV resulted in a mean increase (~4%) in muscle water content (145). The rate of protein degradation was also measured in vitro (145). After 18 hours of MV two strips from the costal diaphragm were removed and suspended in separate in vitro tissue chambers filled with a modified Krebs solution and aerated with 95% O 2 / 5% CO 2 for 2 hours. The tyrosine concentration in the bathing medium was subsequently analyzed fluorometrically (172). The rate of tyrosine release was used to determine the rate of total protein catabolism because this amino acid is neither synthesized nor degraded by skeletal muscle (164). The significant loss of protein, was due in part to the

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37 significant increase in protein degradation, as indicated by a 28% increase in tyrosine release (145). Our results also revealed that MV results in an increase in diaphragmatic oxidative stress (145). The diaphragmatic content of both total 8-isoprostane and protein carbonyls increased 30% and 35% respectively (145). Tissue levels of total 8-isoprostane and protein carbonyls were measured as markers of lipid peroxidation and protein oxidation, respectively. In the context of MV-induced diaphragmatic atrophy, an increase in protein oxidation could be important because moderately oxidized proteins are more sensitive to proteolytic attack by proteases (37, 38, 40, 96, 97, 118). Therefore, oxidative modification of proteins could contribute to the elevated protein degradation measured after MV. Further, the ubiquitin-proteosome pathway is the proteolytic pathway implicated in the degradation of actin and myosin in muscle (55, 158) and this pathway is up-regulated during periods of oxidative stress (142, 146, 161). An oxidative stress-mediated up-regulation of the ubiquitin-proteosome pathway would lead to an increase in protein degradation and thus atrophy. Summary of Literature Review MV is used to sustain patients with a life threatening illnesses, provide ventilation during respiratory muscle failure (respiratory muscle rest) or to compensate for a damaged upper airway or to aid during recovery or rehabilitation from an illness. Excessive inspiratory muscle workload due to obesity, asthma, or pulmonary resection can lead to respiratory muscle failure and require MV to maintain the life of the patient. There are many modes of MV but controlled MV (CMV) results in complete diaphragmatic inactivity and may be an ideal model to study the effects of MV. While CMV provides the maximum degree of respiratory muscle rest it is not without

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38 consequence to the very muscle it is intended to aid, the diaphragm. The reduced use of the diaphragm while receiving MV may lead to atrophy and increase the likelihood of diaphragmatic weakness. If the weakness is substantial it may be difficult to wean the patient. The process of weaning is challenging and contributes to a large portion of the ICU workload. The diaphragm is the primary muscle of inspiration and is chronically active throughout life. The fiber type composition and the metabolic properties of the diaphragm reflect the demand of this chronically active tissue. Studying the diaphragm is often difficult to do using humans but the rat diaphragm has characteristics similar to the human diaphragm, thus enabling difficult research questions to be addressed using this model. The diaphragm, like all skeletal muscle, is highly plastic and therefore rapidly adapts to its demand. Skeletal muscle hypertrophies when muscular activity increases, whereas inactivity or disuse leads atrophy. Hypertrophy and atrophy, are therefore, critically dependent on the relative rates of protein synthesis and protein degradation. Atrophy results in a decrease in CSA and is functionally significant because muscle strength is directly related to CSA. A loss or decrease in neural stimulation of a muscle results in atrophy. There are various models used in the study of hindlimb muscle atrophy (e.g., HI, spaceflight and HU, and ST). Postural locomotor muscles rapidly adapt to the reduced load. Markers of this adaptation include a loss of mass and CSA, slow-to-fast shift in MHC, and a metabolic shift toward a more glycolytic fiber. The functional significance of these adaptations is a loss of strength and endurance.

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39 Manipulation of the phrenic nerve via denervation (phrenicectomy), tetrodotoxin, or spinal cord isolation initially results in diaphragmatic hypertrophy followed by atrophy. Paralyzing the diaphragm results in a significant decrease in specific tension during all stages of its adaptation to paralysis. Attenuation of the contractile and biochemical changes when the inactivity of the muscle fibers is matched to the inactivity of the phrenic motoneurons, spinal cord isolation model, is an intriguing finding. MV, in particular controlled MV, eliminates diaphragmatic EMG activity. The loss of diaphragmatic electrical activity leads to a decrease in force generation and impairs endurance. Specifically, MV leads to a loss of both in vivo and in vitro force generating ability. The impairment in force generation is exacerbated by time spent on the ventilator. The significant loss of diaphragmatic mass (i.e., atrophy) occurs in as few as 18 hours. The loss of diaphragmatic mass includes the loss of both total and myofibrillar protein and this is due in part to an increase in proteolysis.

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CHAPTER 3 METHODS The methods section is organized according to the objectives of each specific aim, animals and experimental design, and the methods used. Experimental Design-Specific Aim #1 The effect of MV on protein synthesis was investigated by testing the hypothesis that MV-induced diaphragmatic atrophy is due, at least in part, to a decreased rate of total (mixed muscle protein (MMP)) and myosin heavy chain (MHC) protein synthesis. Preliminary experiments indicated that prolonged MV results in significant diaphragmatic atrophy. Both MMP and MHC protein synthesis rates (in vivo) in the diaphragms of control, spontaneously breathing, and MV animals during 0 to 6, 6 to 12, and 12 to 18 hours of MV were measured to determine the time course of changes in protein synthesis. Animals and Experimental Design To address this specific aim, healthy young adult (4-month-old) female specific-pathogenfree (SPF) Sprague-Dawley rats were individually housed and fed rat chow and water ad libitum while being maintained on a 12 hour light/dark cycle for 3 weeks before initiation of these experiments. Animals were randomly assigned to either the control or MV experimental group. The control group was subdivided into four groups, control, or 6 to 18 hours of spontaneously breathing (SB 6, SB 12, SB 18). The control animals (n = 10) were not mechanically ventilated nor did they receive infusion of [113 C]leucine. This group was used to determine the natural abundance of [113 C]leucine in the Sprague40

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41 Dawley rat diaphragm. The SB 6 group (n = 10) was not mechanically ventilated but they were infused with [113 C]leucine while spontaneously breathing and anesthetized for 6 hours. Animals assigned to the SB 12 group (n = 10) were anesthetized for a total of 12 hours and [113 C]leucine was infused during hours 6 to12. Similarly, the SB 18 group (n = 10) was anesthetized for a total of 18 hours and [113 C]leucine was infused during hours 12 to 18. The SB groups served as time matched controls for the MV groups. The MV group was subdivided into three groups, MV 6, MV 12, and MV 18. The MV groups were mechanically ventilated and the rate of protein synthesis was determined after 6 (n = 10), 12 (n = 10), and 18 (n = 10) hours of MV. [113 C]leucine was infused during the last 6 hours of the experimental period (i.e., 0 to 6, 6 to 12, and 12 to 18 hours). Control (n=10)no MV (acute) no c13Leu SB 6 (n=10)no MVc13Leu infusionduring hrs 0-6 SB 12 (n=10)no MVc13 Leu infusion during hrs 6-12 SB 18 (n=10)no MVc13 Leu infusionduring hrs 12-18 MV 6 (n=10).c13Leu infusionduring hrs 0-6 MV 12 (n=10).c13Leu infusionduring hrs 6-12 MV 18 (n=10).c13Leu infusionduring hrs 12-18 ExperimentalDesign Measure [113 C]leucine incorporation Figure 3-1. Experimental design for Specific Aim #1. Mechanical ventilation protocol Thirty minutes before anesthesia, animals received glycopryyloate (0.04 mg/kg IM) in order to reduce airway secretions. Glycopryyolate was then administered every 2 hours (0.04 mg/kg IM) for the remainder of the experiment. The animals were anesthetized

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42 with an intraperitoneal (IP) injection of sodium pentobarbital (50 mg/kg of body weight). Sodium pentobarbital was used as the general anesthetic because Le Bourdelles et al. (92) have shown that the level of barbiturate required to maintain general anesthesia in rats does not alter locomotor muscle contractile or biochemical properties. Additionally, prolonged use of sodium pentobarbital (up to 18 hours) in rats does not induce atrophy in locomotor muscles (i.e., soleus) (145). All surgical procedures were performed under aseptic conditions. Once anesthetized, MV animals were tracheostomized and mechanically ventilated with a volume-cycled ventilator (Inspira, Harvard Apparatus, Cambridge, MA). A major advantage of volume-cycled ventilators is that tidal volume remains relatively constant despite possible pathophysiological changes (e.g., airway obstruction due to mucus secretion). Further, volume-cycled ventilators are capable of maintaining a constant inspired percentage of oxygen (F I O 2 ) which is important in maintaining blood gas homeostasis during MV (20, 72, 165). Heart rate and electrical activity of the heart was monitored via a lead II ECG using needle electrodes placed subcutaneously. An arterial catheter was placed in the carotid artery for constant measurement of blood pressure and blood samples (1 mL) were drawn for analysis of [113 C]leucine. Finally, a venous catheter was placed in the jugular vein to add fluids and sodium pentobarbital. Anesthesia was maintained during MV by intravenous infusion of sodium pentobarbital (10 mg/kg body weight). Body (rectal) temperature was maintained at 37C 1C by use of a computer controlled re-circulating heating blanket. Throughout MV body fluid homeostasis was maintained via the administration of an intravenous electrolyte solution, 2 mL/kg/hour. Continuing care during MV included expressing the

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43 bladder, removal of airway mucus, lubricating the eyes, rotating the animal, and passive movement of the limbs. Animals were continuously monitored during MV and while spontaneously breathing, see below. Postmortem examination A board-certified pathologist (Dr. Sunjoo Kim, M.D.) performed all postmortem procedures. Dr. Kim has extensive necropsy experience and is experienced in the clinical microbiology techniques used to assess pneumonia and septicemia. Necropsy examination. Necropsy examination included a detailed visual inspection of the respiratory tract, the lungs, and the peritoneal cavity. Visualization of an abscess or pus was considered a marker of infection, however no animals were infected. Blood culture. Blood culture procedures were preformed according to the methods recommended by the American Society for Microbiology (21) and Bailey and Scotts Diagnostic Microbiology (82). A blood sample (0.5 mL) was drawn from each animal at the conclusion of the experimental period via the jugular vein. Each sample was immediately inoculated directly into the MacConkey agar plate (82). The blood cultures were incubated at 37C for 5 days and inspected daily (82). Observation of bacterial growth on the culture media would have been considered evidence of sepsis. Control animals (nonmechanically ventilated) protocol The animals in the control groups were randomly placed into one of four groups: Control (acute anesthesia, no MV, no [113 C]leucine infusion) or SB 6, SB 12, or SB 18 (anesthetized, spontaneously breathing, [113 C]leucine infusion). Control animals (acute anesthesia) were free of intervention during the hours before removal of the diaphragm for measurement of biochemical properties. That is, these animals were not mechanically ventilated before study. Control animals received an IP injection of sodium pentobarbital

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44 (50 mg/kg body weight); after a surgical plane of anesthesia was reached their diaphragms were removed for subsequent measurements of biochemical properties. SB 6, SB 12, and SB 18 animals received the same surgical intervention and [113 C]leucine infusion paradigm as the MV animals except these animals were not mechanically ventilated (i.e., they were breathing on their own during the entire experimental period). The MV and SB animals received the same continuing care during the experimental period and post mortem examination after the experimental period. Methods Used: Biochemical Assays Tissue removal and storage At the appropriate times (as specified in the experimental design) biochemical studies were conducted on muscle samples taken from the costal portion of the diaphragm. Costal diaphragm segments obtained for biochemical analysis were rapidly frozen in liquid nitrogen and stored at C until assay. Rates of in vivo diaphragmatic protein synthesis Infusion The fractional rate of diaphragm muscle protein synthesis was measured using intravenous infusion of [113 C]leucine (Cambridge Isotopes Laboratory, Andover, MA) and quantifying the in vivo rate of incorporation of [113 C]leucine into both total and contractile proteins in the diaphragm by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The stable isotope [113 C]leucine was chosen for several reasons: Less isotope effect (i.e., less tissue injury). Small tissue sample is required for analysis. Safety. Validity and reliability of this label for measurement of the rate of in vivo muscle protein synthesis is well established (121).

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45 Animals were anesthetized with sodium pentobarbital and a tygon catheter was placed in the jugular vein for infusion of [113 C]leucine. A polyethylene catheter was also placed in the carotid artery for blood sampling. Note, the jugular and carotid catheters were the same as described above for adding electrolyte solution and sodium pentobarbital and for monitoring arterial blood pressure. The jugular vein and carotid artery were catheterized in the SB and MV groups immediately after anesthetization. The catheter was kept patent by periodically flushing with heparinized saline (20 U/mL). At the beginning of the infusion, animals were primed with 1.6 mg [113 C]leucine/100 grams of body weight, followed by a constant infusion rate of 0.20 mg[113 C]leucine/100 grams of body weight/ hour for 6 hours (KD Scientific Model 100 syringe pump, Boston, MA). This infusion rate was chosen on the basis of previous experiments indicating that this rate results in optimal muscle [113 C]leucine enrichment for the determination of protein synthesis in rat skeletal muscle (181). The infusion of the labeled amino acid is delivered such that ~5-10% of the plasma pool of free amino acids is labeled (130). In this sense the labeled amino acid acts as a tracer with little or no effect on the overall metabolism of tissue being investigated (130). The duration of the infusion period need only be long enough for enrichment of the target protein but not so long as to allow recycling of the tracer (130). Skeletal muscle protein turns over slowly and thus tracer recycling does not present a problem (130). Immediately before infusion, and at the end of the 5 th and 6 th hours of infusion, blood samples (1 mL) were drawn for the measurement of plasma -ketoisocaproate (-KIC) enrichment. At the completion of the experimental period, the costal diaphragm was rapidly removed, frozen in liquid nitrogen, and stored at C until assay. Note that the

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46 infusion pump continued to infuse at the time of tissue removal. A tissue sample only needs to be taken at the end of the infusion period if no other labeled amino acid has been administered previously. The baseline sample labeling can be assumed to be close to previously measured tissue from the same tissue population (57) or from mixed blood protein (73). Sample analysis and calculations Plasma -KIC was isolated, prepared as the trimethylsilyl quinoxalinol derivative, and analyzed for [113 C]leucine abundance by use of gas chromatography-electron impact quadrupole-mass spectrometry (GC-MS) (HP 5890 Series II and GC/HP 5970 Series MS, Hewlett-Packard, Avondale, PA). Tissue fluid-free amino acids were extracted by homogenization in 10% TCA. The N-heptafluorobutyryl propyl esters were formed, and the [113 C]leucine abundance was determined using electron capture-negative chemical ionization GC/MS (HP 5988A Series II and GC/HP 5970 Series MS, Hewlett-Packard). The plasma -KIC enrichment and the tissue fluid [113 C]leucine enrichment [in mole percent in excess (MPE)] were used to represent the precursor pool (leucyl tRNA) enrichment for the calculation of protein synthesis rate (K s ; the percentage of the protein mass synthesized per hour (%/hour)). The following equation was used to calculate K s )t-(t enrichemnt poolprecursor 100 protein in enrichment MPE leucine 13C]-[112 Ks where (t 2 -t 1 ) is the infusion time (hours). The rate of protein synthesis can be determined if the site of the incorporation of the amino acid into protein (i.e., in the appropriate aminoacyl-tRNA pool, is known) (130). However since identification of the correct pool presents a problem (130), a surrogate index of the labeling of the aminoacyl-tRNA pool has been adopted. The

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47 branched chain amino acid leucine is the preferred tracer and the labeling of its transamination product, -ketoisocaproate (-KIC), is measured. The use of -KIC as the surrogate of the true precursor labeling is possible because the formation of its transamination product, -KIC, occurs intracellularly and thus -KIC reflects the extent of labeling in the true precursor pools for protein synthesis (130). Indeed, the labeling of -KIC after infusion of labeled leucine is within 10% of the labeling of tRNA in human skeletal muscle (174). Analysis of mixed muscle protein synthesis rates To determine the [113 C]leucine abundance in mixed muscle protein (MMP) 50-60 mg muscle samples were homogenized in 1 mL of 10% TCA and hydrolyzed in 6 N HCl at 110C for 24 hours. The n-acetyl n-propyl (NAP) esters of the component amino acids were formed, and the [113 C]leucine abundance in the hydrolyzed MMP were determined using GC-C-IRMS according Yarasheski et al. (181). Isolation of MHC for analysis of synthesis rates All procedures for the MHC extractions were performed on ice or at 4C. Frozen muscle samples (60-80 mg) were homogenized in 1 mL of a 250 mM sucrose buffer (in mM: 250 sucrose, 100 KCl, 5 EDTA, and 20 imidazole, pH 6.8). The homogenate was centrifuged at 1,200 x g for 10 minutes, and the supernatant was discarded. The pellet was suspended in 1 mL of a 0.5% Triton X-100 solution (175 mM KCl, 0.5% Triton X-100, pH 6.8), a modification of Solaro et al. (150). The suspension was homogenized and centrifuged as before. This step removes many of the soluble matrix proteins. The resultant pellet was rinsed (i.e., homogenized and centrifuged) with 1 mL wash buffer (150 mM KCl and 20 mM Tris, pH 7.0) to remove excess Triton X-100 solution. The pellet was then frozen at C. The

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48 pellet was resuspended in SDS buffer (62.5 mM Tris, 2% SDS, 10% glycerol, 0.001% bromophenol blue, and 5% -mercaptoethanol) and boiled for 5 minutes. These extracts of myofibrillar protein (containing ~1 mg) were separated by SDS-PAGE. All electrophoresis chemicals were purchased from Bio-Rad (Hecules, CA). Each extract was separated on an individual gel with a single wide lane, utilizing a 7% T-2.5% C polyacrylamide slab gel with a 4% T-2.5% C stacking gel. The proteins were separated (150 volts, ~5 hours) using a discontinuous buffer system that useed a Tris-Tricine buffer (1.6 mM Tris, 16 mM Tricine, 0.01% SDS, pH 6.4) and a Tris buffer (2.5 mM Tris, 0.01% SDS, pH 6.4) as the cathode and anode buffers, respectively. The separated protein was visualized by Coomassie staining (0.1% Coomassie brilliant blue R-250, 45% methanol, and 10% glacial acetic acid) for 10 to 15 minutes. after overnight destaining (30% methanol and 10% glacial acetic acid), the band corresponding to the molecular mass of MHC was identified. The protein band was carefully cut out within the distinctly stained boundary, minced, and put into a tube. The samples were hydrolyzed in 3 to 4 mL of concentrated HCl (110C for 48 hours), and the NAP esters of the amino acids were prepared for analysis of [113 C]leucine abundance using GC-C-IRMS. Measuring mixed muscle (MMP) and MHC protein synthesis is very involved but the method is well established and in everyday use in the Yarasheski laboratory. The method of accurate identification of the MHC band, the quantity and quality of the band, and the reliability of the isotopic enrichments are well documented (70).

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49 Statistical analysis Planned comparisons were made between relevant groups with a Bonferroni correction for the number of comparisons conducted. Significance was established at p < 0.05. Experimental Design-Specific Aim #2 The effect of MV on MHC mRNA content was determined by testing the hypothesis that MV alters pretranslational events in the diaphragm. Preliminary experiments indicated that prolonged MV results in significant diaphragmatic atrophy and the proposed experiments determined the time course of changes in MHC mRNA during 6 to 18 hours of MV. To test our hypothesis we measured type I, IIa, IId/x, and IIb MHC mRNA content in diaphragms from control, SB, and MV animals after 6, 12, and 18 hours of SB or MV. The measurements were made using a portion of the costal diaphragm from the same group of animals used to test the first hypothesis. The experimental design, surgical procedures, care, diaphragm removal, necropsy examination, and blood culture have been described in Specific Aim #1. Methods Used: Biochemical Assays Total RNA isolation A portion of the costal diaphragm, ~50 mg, was homogenized in 1.5 mL of Trizol (Invitrogen, Carlsbad, CA) and processed according to the manufactures instructions. This protocol is based on the method described by Chomczynski and Sacchi (34). The sample was centrifuged at 12,000 x g for 10 minutes to the remove insoluble material. The RNA portion, the upper aqueous phase, was transferred and incubated at room temperature for 5 minutes. The RNA was extracted with bromochloropropane, precipitated with isopropanol, washed with 75% ethanol, and pelleted via centrifugation.

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50 The pellet was resuspended in RNAse free water (Sigma, St. Louis, MO). The concentration and purity of the total RNA extracted was measured spectrophotometrically at 260 nm and at 280 nm in 1x TE buffer (Promega, Madison, WI). Ideally, the ratio of A 260 /A 280 should be greater than 1.8. This is a measure of RNA purity. Absorbance at 260 nm (A 260 ) reflects the RNA contration and absorbance at 280 nm (A 280 ) reflects the protein content. The concentration of RNA was determined via the equation: 101 read of #40 260at OD/ LLgRNA The optical density (OD) of RNA at 260 nm (OD 260 ) is assumed to equal 40 g/L (178). This method yields un-degraded RNA, free of DNA and proteins. The integrity of the extracted total RNA was verified by gel electrophoresis of 1 g RNA on an 1% agarose tri(hydroxymethyl)aminomethane (Tris)-borate-EDTA buffer (TBE) gel containing ethidium bromide using 1x TBE as the running buffer. Both the 28S and 18S (ribosomal RNA) bands were clear and distinct in the intact samples. Samples that did not demonstrate these characteristics (i.e., degraded samples) were discarded and another tissue sample from the same animal was processed and an un-degraded RNA sample was then analyzed. The total RNA samples were then stored at 80C until analysis. Reverse transcription (RT) Total RNA was reverse transcribed and amplified via PCR for each diaphragm sample. Briefly, 1 g of total RNA was reverse transcribed using SuperScript II RT (Invitrogen, Carlsbad, CA) and a mix of oligo dT (100 ng/reaction) and random primers (200 ng/reaction) in a final volume of 20 L according to the protocol provided by the

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51 manufacturer. An equal number of samples from each group were included in each run. The samples were then stored at C until used for PCR. Polymerase chain reaction (PCR) MHC content was determined via relative RT-PCR with 18S serving as the internal standard (Ambion, Austin TX). The MHC primer sequences used have been published by Dr. Ken Baldwins group (178) and are shown in Table 3-1 The primer for the five prime (5) end of each mRNA was designed from a highly conserved region in all known rat MHC genes ~600 base pairs upstream of the stop codon (99). The four adult rat MHC isoforms (I, IIa, IId/x, and IIb) are identical in this region. This allowed for the design of a common primer with the following sequence:5 GAA GGC CAA GAA GGC CAT C 3 (178). The primers for the three prime (3) end were derived from the 3untranslated regions (UTR) of each of the different MHC genes, the sequences for each rat MHC gene are highly specific in this region (42, 65, 66). In order to account for differences in the initial amount of total RNA and to serve as an internal standard, 18S ribosomal RNA was co-amplified with the target cDNA (mRNA) in each PCR sample. The Alternate 18S Internal Standard kit (Ambion, Austin, TX) was used. The 18S primers were mixed with the provided competimer in a 1:4 ratio. The 18S competimer is required to decrease the 18S signal. The 18S primer to competimer ratio was optimized such that amplification of the target cDNA and 18S ribosomal RNA was similar (Ambion, Relative RT-PCR kit protocol). The PCR conditions were as follows: 2 mM MgCl 2 in standard PCR buffer (Invitrogen, Carlsbad, CA), 0.2 mM dNTP, 0.2 M of the common primer, 0.2 M of one of the four gene specific primers, 0.5 M 18S primer/competimer mix, 2 L of the

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52 diluted RT product (1 L of each RT reaction was diluted 40 fold before PCR amplification), and 0.75 units of Taq polymerase (Invitrogen, Carlsbad, CA) in a final volume of 25 L. All four MHC genes were amplified in four separate reactions for each experimental group and one sample form each experimental group was included in each PCR run. PCR was carried out with an initial 3 minute denaturation step at 96C, followed by 24 cycles, each cycle consisting of 45 seconds at 96C (denaturation), 60 seconds at 50C (primer annealing), 90 seconds at 72C (extension), and a final step of 3 minutes at 72C using the Stratagene Robocycler. The number of cycles was determined to be on the linear portion of a semilog plot of the yield (see below) expressed as a function of cycle number. The PCR products were separated by agarose gel electrophoresis [20 L sample of the PCR product loaded on 2.0% agarose gels (in 1x TBE buffer) containing 0.2 g/mL ethidium bromide] for visualization of the PCR products. Analysis of gels A digital image of each gel was captured and the bands were analyzed via computerized densitometry (Gel-Doc 2000, Bio-Rad, Hercules, CA). The integrated areas of each target (MHC) and 18S internal control fragment DNA band were determined with the local background subtracted. The integrated area of the target band was normalized to the integrated area of the corresponding 18S internal control fragment, thus correcting for any differences in PCR reaction efficiencies. The value for each MHC mRNA is expressed as MHC mRNA/18S.

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53 Statistical analysis Planned Comparisons were made between relevant groups with a Bonferroni correction for the number of comparisons conducted. Significance was established at p < 0.05. Table 3-1. Oligonucleotide primers used for the PCR reactions MRNA Common Primer (5 end) Antisense Primer (3 end) Sample cDNA Type I MHC 5 GAA GGC CAA GAA GGC CAT C 3 5 GGT CTC AGG GCT TCA CAG GC 3 596 bp Type IIa MHC 5 GAA GGC CAA GAA GGC CAT C 3 5 TCT ACA GCA TCA GAG CTG CC 3 570 bp Type IId/x MHC 5 GAA GGC CAA GAA GGC CAT C 3 5 GGT CAC TTT CCT GCT TTG GA 3 574 bp Type IIb MHC 5 GAA GGC CAA GAA GGC CAT C 3 5 GTG TGA TTT CTT CTG TCA CC 3 590 bp Sample cDNA is the size of the myosin heavy chain (MHC) mRNA PCR product in base pairs (bp). common stop codon p rime r region ~600 bp MHC sequences 3 UTR Figure 3-2. Schematic representation of the myosin heavy chain (MHC) genes, the common primer is identical in all sequences, followed by ~600 base pairs (bp) of coding sequence. A stop codon and 3-untranslated region (UTR) that are highly specific for each MHC gene with little or no sequence similarity among gene family members are depicted.

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CHAPTER 4 RESULTS Morphological, Physiological, and Post Mortem Observations The body mass characteristics of each experimental group are presented in Table 41 No significant differences (p > 0.05) existed in pre-experiment or post-experiment body mass between groups. Importantly, no group experienced a significant (p > 0.05) loss of body mass over the course of the experiment, indicating adequate hydration during the experimental period. Additionally, all animals urinated and experienced intestinal transit during the experimental period. Heart rate and systolic blood pressure were monitored as a means of determining animal homeostasis during the experimental period. The mean heart rate and systolic blood pressure data are presented in Tables 4-2 and 4-3 respectively. Heart rate and blood pressure were within normal ranges and were well maintained during the experiments. Further, note that the heart rate and blood pressure responses were very similar between the SB animals and the MV animals at each time point. Post mortem examination of the SB and MV animals included a necropsy and blood culture. No animals demonstrated any signs of infection, weight loss, or post mortem abnormalities, and all blood cultures were negative for bacteria. Additionally, the colonic temperature of each animal remained constant, 37 0.5C, during the experiments. Collectively, these results indicate that our aseptic surgical technique successfully prevented infection. 54

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55 Influence of Mechanical Ventilation on Protein Synthesis MV resulted in depressed protein synthesis in the diaphragm within the first 6 hours; this reduced rate of protein synthesis persisted throughout the remainder of the experimental period. Note that within the first 6 hours, the rate of both mixed muscle protein (MMP) (-30%) and MHC (-65%) protein synthesis significantly decreased (p < 0.05). Precursor pool 13 C enrichment, MMP and MHC 13 C enrichment, and MMP and MHC fractional synthetic rates are each presented in Figures 4-1 4-2 and 4-3 Enrichment values are expressed as mole percent in excess (MPE). Mole percent in excess is the enrichment of 13 C above natural levels. Plasma enrichment of 13 C in MPE was determined from the baseline plasma sample in each animal before infusion. Tissue fluid, MMP, and MHC 13 C enrichment in MPE was calculated using the acute anesthesia animals as the baseline measure of naturally occurring levels of 13 C. Figure 4-1 reports plasma [ 13 C]leucine and [113 C]ketoisocaproic acid ([ 13 C]KIC) enrichment. The plateau in the enrichment of the plasma precursor pools indicates that a steady-state was achieved by the 6 th hour of infusion. No significant differences (p > 0.05) existed in the enrichment of plasma [ 13 C]leucine or [ 13 C]KIC after 5 hours of infusion compared to 6 hours of infusion within groups (e.g., SB 6 at hour 5 vs. hour 6). Additionally, the enrichment of plasma [ 13 C]leucine or [ 13 C]KIC did not differ between groups (e.g., SB 6 vs. MV 6). Rates of protein synthesis calculated using the plasma [ 13 C]leucine and [ 13 C]KIC precursor pools were made using the 6 hour enrichment values. The [ 13 C]leucine enrichment of the tissue fluid precursor pool did not differ between time matched groups ( Figure 4-2 ). Note that the 18 hour SB and MV groups

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56 each experienced significantly greater (p < 0.05) tissue fluid [ 13 C]leucine enrichment than did their 6 and 12 hour counterparts. By averaging the endogenous amount of 13 C in the diaphragms from the Control acute anesthesia group and subtracting the amount of endogenous 13 C from all SB and MV values we derived the degree of 13 C enrichment of the diaphragm proteins. The difference between the endogenous 13 C content and the measured 13 C after [ 13 C]leucine infusion is termed the degree of enrichment. The SB group experienced a significant (20%, p < 0.05) decrease in diaphragmatic MMP [ 13 C]leucine enrichment over time (hour 6 to hour18). In comparison to their time matched counterparts, the MV animals experienced a significant (30-34%, p < 0.05) decrease in diaphragmatic MMP [ 13 C]leucine enrichment ( Figure 4-3 ). [ 13 C]leucine enrichment of MHC protein in the diaphragm remained constant over time in the SB group. A significant (68 to 75%, p < 0.05) decrease in the enrichment of diaphragm MHC protein was observed during MV at each time point ( Figure 4-3 ). After measurement of precursor pool (plasma and tissue fluid) and MMP and MHC enrichment, calculation of the fractional synthetic rate of protein synthesis was performed. The fractional synthetic rate of both MMP and MHC protein was calculated using all three surrogates of [ 13 C]leucy-tRNA: plasma [ 13 C]leucine, plasma [ 13 C]KIC, and tissue fluid [ 13 C]leucine. MV significantly (p < 0.05) slowed the fractional synthetic rate of both MMP and MHC protein synthesis ( Figure 4-4 Figure 4-5 and Table 4-4 ). Regardless of which precursor pool was used for these calculations the decreased fractional synthetic rate remained. However, for clarity, only the fractional synthetic rate

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57 calculated using the tissue fluid [ 13 C]leucine precursor pool is presented in graphic form ( Figure 4-4 and Figure 4-5 ). Mixed muscle protein synthesis, a measure of whole muscle protein synthesis, slowed significantly (30%, p < 0.05) during the first 6 hours of MV as compared to the time matched SB 6 group ( Figure 4-4 and Table 4-4 ). The 30% decrease in the rate of MMP synthesis persisted at hour 12 (MV 12 compared to SB 12, -26%) and hour 18 (MV 18 compared to SB 18, -29%). Myosin heavy chain protein synthesis was measured in order to estimate the impact of MV of the rate of contractile protein synthesis. Within the first 6 hours of MV, MHC protein synthesis slowed significantly (66%, p < 0.05) as compared to the time matched SB 6 group ( Figure 4-5 and Table 4-4 ). In parallel with the MMP synthesis rates, the decrease in MHC protein synthesis rates after MV remained constant as compared to each time matched SB group. Total RNA and Myosin Heavy Chain mRNA in the Diaphragm after Spontaneous Breathing and Mechanical Ventilation Total RNA is ~80-85% ribosomal RNA (rRNA) and can be used as an index of the quantity of ribosomal subunits and as an indirect index of the synthetic capacity of the tissue. In contrast, mRNA constitutes ~2-3% of the total RNA pool. Total RNA was isolated from each diaphragm and the mRNA encoding the four adult MHC phenotypes was then measured to determine if the observed decrease in protein synthesis was due, in part, to a decrease in total RNA and/or MHC mRNA. The total RNA findings are presented in Table 4-5 Exposure to the anesthetic (SB groups) or MV did not affect the amount of total RNA isolated from the diaphragms of any of the groups.

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58 Once isolated from the diaphragm, total RNA was reverse transcribed. The cDNA and the 18S rRNA control fragment were then amplified via PCR and the products were separated electrophoretically on 2% agarose gels stained with ethidium bromide. An example of the products is depicted in Figure 4-6 In each lane (top to bottom) the two products, the amplified target mRNA and the amplified 18S internal standard fragment, were present. The upper band is the amplified target mRNA and the lower band is the 18S rRNA internal standard fragment. The expected size of each amplified target mRNA product is 596 bp, 570 bp, 574 bp, 590 bp for type I, IIa, IIx, and IIb MHC, respectively. The expected 18S control fragment size is 324 bp. Figure 4-7 through Figure 4-10 shows MHC mRNA results. The MHC mRNA data are expressed relative to the 18S rRNA internal standard product to account for variations in the RT-PCR process. Exposure to prolonged anesthesia (SB) or MV did not alter the relative amount of any of the four MHC mRNAs. The graphic results for each MHC mRNA are depicted in Figure 4-7 through Figure 4-10 Table 4-1. Animal body mass before and after experimental period Group Body Mass (g) before Body Mass (g) after Control 255.0 6.8 ---------SB 6 251.9 3.6 251.7 3.6 MV 6 248.5 4.2 248.5 4.2 SB 12 259.7 3.0 258.3 3.0 MV 12 262.8 2.5 262.7 2.3 SB 18 259.0 3.8 259.4 4.2 MV 18 259.7 3.1 259.4 3.3 Values are means standard error (SE) with n = 10 per group. SB = spontaneously breathing. MV = mechanically ventilated.

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59 Table 4-2. Heart rate response during MV and SB Group Time zero 6 th hour 12 th hour 18 th hour Control N/D SB 6 343 9 352 6 MV 6 357 3 360 6 SB 12 351 5 361 1 350 10 MV 12 375 7 358 14 364 6 SB 18 340 12 360 11 324 322 22 MV 18 374 10 363 5 388 11 395 17 Values are means SE expressed in beats per minute with n = 10 per group. N/D = no data. SB = spontaneously breathing. MV = mechanically ventilated. Table 4-3. Systolic blood pressure response during MV and SB Group Time zero 6 th hour 12 th hour 18 th hour Control N/D SB 6 135 4 109 5 MV 6 122 3 108 5 SB 12 137 3 129 6 103 7 MV 12 154 6 107 106 8 SB 18 128 5 112 5 96 5 92 3 MV 18 138 3 110 5 100 8 98 7 Values are means SE expressed in mmHg with n = 10 per group. N/D = no data. SB = spontaneously breathing. MV = mechanically ventilated.

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60 Tracer Infusion Time (hr) 456 Plasma 13CMole % Excess 0510152025 SB 6 MV 6 SB 12 MV 12 SB 18 MV 18 13C-Leucine13C-KIC Figure 4-1. Plasma [ 13 C]leucine and plasma [ 13 C]ketoisocaproic acid ([ 13 C]KIC) enrichment. Values are means SE expressed in mole percent in excess of natural 13 C abundance with n = 10 per group. SB = spontaneously breathing animals; MV = mechanically ventilated animals. Hour 5 = plasma [ 13 C]leucine or [ 13 C]KIC after 5 hours of infusion; hour 6 = plasma [ 13 C]leucine or [ 13 C]KIC after 6 hours of infusion.

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61 SB 6MV 6SB 12MV 12SB 18MV 18 Mole % Excess 0246810121416 ** Figure 4-2. Tissue fluid [ 13 C]leucine enrichment in the diaphragm. Values are means SE expressed in mole percent in excess of natural 13 C abundance measured in Control animal diaphragm with n=10 per group. SB = spontaneously breathing animals; MV = mechanically ventilated animals. Significantly different (p < 0.05) from SB 18; significantly different (p < 0.05) from MV 18.

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62 SB 6MV 6SB 12MV 12SB 18MV 18 Mole % Excess 0.000.020.040.060.080.100.120.140.160.18 MMP MHC ****** Figure 4-3. Mixed muscle protein and myosin heavy chain [ 13 C]leucine enrichment in the diaphragm. Values are means SE expressed in mole percent in excess of natural 13 C abundance measured in Control animal diaphragm with n=10 per group. MMP= mixed muscle protein; MHC = myosin heavy chain; SB = spontaneously breathing animals; MV = mechanically ventilated animals. Significantly different (p < 0.05) from time matched SB; significantly different (p < 0.05) from SB 6, significantly different (p < 0.05) from MV 6.

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63 SB 6MV 6SB 12MV 12SB 18MV 18 %/hr 0.000.050.100.150.200.250.30 *** Figure 4-4. Fractional synthetic rates of mixed muscle protein (MMP) by calculation with tissue fluid [ 13 C]leucine as the surrogate measure of the [ 13 C]leucyl-tRNA precursor pool. Values are means SE expressed in percent per hour (%/hr) with n=10 per group. SB = spontaneously breathing animals; MV = mechanically ventilated animals. Significantly different (p<0.05) from time matched SB group; significantly different (p < 0.05) from SB6.

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64 SB 6MV 6SB 12MV 12SB 18MV 18 %/hr 0.000.020.040.060.080.100.120.14 *** Figure 4-5. Fractional synthetic rates of myosin heavy chain (MHC) protein by calculation with tissue fluid [ 13 C]leucine as the surrogate measure of the [ 13 C]leucyl-tRNA precursor pool. Values are means SE expressed in percent per hour (%/hr) with n=10 per group. SB = spontaneously breathing animals; MV = mechanically ventilated animals. Significantly different (p<0.05) from time matched SB group; significantly different (p < 0.05) from SB 6.

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65 Table 4-4. Fractional synthetic rates of mixed muscle protein and myosin heavy chain protein by calculation with each surrogate of the [ 13 C]leucyl-tRNA precursor pool. Plasma [ 13 C]leucine (%/hr) Group MMP MHC 6 hour SB 0.1381 0.0077 0.0514 0.0068 6 hour MV 0.0886 0.0077 0.0178 0.0056 12 hour SB 0.1013 0.0044 0.0487 0.0088 12 hour MV 0.0624 0.0066 0.0173 0.0104 18 hour SB 0.1174 0.0055 0.0419 0.0056 18 hour MV 0.0813 0.0055 0.0118 0.0027 Plasma [ 13 C]KIC (%/hr) Group MMP MHC 6 hour SB 0.2454 0.0127 0.0920 0.0132 6 hour MV 0.1747 0.0110 0.0321 0.0088 12 hour SB 0.1827 0.0077 0.0864 0.0121 12 hour MV 0.1281 0.0110 0.0304 0.0176* 18 hour SB 0.2277 0.0110 0.0774 0.0099 18 hour MV 0.1466 0.0110* 0.0225 0.0051*

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66 Table 4-4. (continued) Tissue Fluid [ 13 C]leucine (%/hr) Group MMP MHC 6 hour SB 0.2040 0.0110 0.0990 0.0180 6 hour MV 0.1691 0.0110 0.0341 0.0092* 12 hour SB 0.1879 0.0088 0.0899 0.0165 12 hour MV 0.1388 0.0088 0.0310 0.01650* 18 hour SB 0.1636 0.0077 0.0584 0.0048 18 hour MV 0.1164 0.0055 0.0187 0.0044* Values are means SE expressed in percent per hour with n= 10 per group. MMP = mixed muscle protein; MHC = myosin heavy chain; SB = spontaneously breathing animals; MV = mechanically ventilated animals. Significantly different (p<0.05) from time matched SB group; significantly different (p < 0.05) from SB 6, significantly different (p < 0.05) from MV 6. Note that the Tissue Fluid [ 13 C]Leucine data are also presented in Figures 4-4 and 4-5 Table 4-5. Total RNA obtained from the costal diaphragm Group Total RNA (g/mg) Control 0.923 0.200 6 hour SB 0.937 0.032 6 hour MV 0.962 0.022 12 hour SB 0.898 0.017 12 hour MV 0.910 0.018 18 hour SB 0.959 0.028 18 hour MV 0.991 0.023 Values are means SE expressed as g of total RNA and as the amount (g) of total RNA per mg wet weight of diaphragm with n = 10 per group. SB = spontaneously breathing; MV = mechanically ventilated.

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67 I IIa C SB6 MV6 SB12 MV12 SB18 MV18 C SB6 MV6 SB12 MV12 SB18 MV18 IIx IIb C SB6 MV6 SB12 MV12 SB18 MV18 C SB6 MV6 SB12 MV12 SB18 MV18 T 18S T 18S MWM (BP) 600 300 600 300 Figure 4-6. RT-PCR products separated on a 2% agarose gel with n=10 per group. C = Control, acute anesthesia animals; SB = spontaneously breathing animals; MV = mechanically ventilated animals; MWM = molecular weight marker in basepairs (bp); T = target MHC mRNA; 18S = 18S rRNA internal standard. ControlSB 6MV 6SB 12MV 12SB 18MV 18 Type I MHC mRNA / 18S 0123 Figure 4-7. Relative type I MHC expression. Values are means SE expressed as type I MHC mRNA normalized to 18S rRNA internal standard (MHC mRNA / 18S) with n=10 per group. C = Control, acute anesthesia animals; SB = spontaneously breathing animals; MV = mechanically ventilated animals.

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68 ControlSB 6MV 6SB 12MV 12SB 18MV 18 Type IIa MHC mRNA / 18S 012 Figure 4-8. Relative type IIa MHC expression. Values are means SE expressed as type IIa MHC mRNA normalized to 18S rRNA internal standard (MHC mRNA / 18S) with n=10 per group. C = Control, acute anesthesia animals; SB = spontaneously breathing animals; MV = mechanically ventilated animals.

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69 ControlSB 6MV 6SB 12MV 12SB 18MV 18 Type IIx MHC mRNA / 18S 012 Figure 4-9. Relative type IIx MHC expression. Values are means SE expressed as type IIx MHC mRNA normalized to 18S rRNA internal standard (MHC mRNA / 18S) with n=10 per group. C = Control, acute anesthesia animals; SB = spontaneously breathing animals; MV = mechanically ventilated animals.

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70 ControlSB 6MV 6SB 12MV 12SB 18MV 18 Type IIb MHC mRNA / 18S 012 Figure 4-10. Relative type IIb MHC expression. Values are means SE expressed as type IIb MHC mRNA normalized to 18S rRNA internal standard (MHC mRNA / 18S) with n=10 per group. C = Control, acute anesthesia animals; SB = spontaneously breathing animals; MV = mechanically ventilated animals.

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CHAPTER 5 DISCUSSION Overview of Principle Findings These experiments investigated the affect of 6 to 18 hours of MV on protein synthesis and MHC mRNA in the rat diaphragm. Our results support the hypothesis that the MV-induced diaphragmatic atrophy is, at least in part, due to a decreased rate of total (MMP) and myosin heavy chain (MHC) protein synthesis. Indeed, within the first 6 hours of MV MMP synthesis decreased by ~30% and MHC protein synthesis decreased by ~65%. These decrements in protein synthesis persisted throughout the 18 hours of MV. In contrast, our data do not support the postulate that MV alters pretranslational events in the diaphragm as indicated by the observation that MV did not alter MHC mRNA content. A detailed discussion of these points follows. Impact of Mechanical Ventilation on Protein Synthesis in the Diaphragm Mixed Muscle Protein Synthesis MMP synthesis is the average synthetic rate of all proteins (e.g., contractile proteins, sarcoplasmic reticulum proteins, enzymatic proteins) in the muscle sample and was measured as an index of total protein anabolism in the diaphragm. The fractional rate of MMP synthesis in the diaphragm was measured after three periods of MV (6, 12, and 18 hours), and compared to time matched controls. The SB 6 MMP synthetic rate calculated using tissue fluid [ 13 C]leucine was ~0.2 %/h. These results are lower than published MMP synthetic rates in the rat diaphragm, ~0.4%/h to 0.6%/h (50, 127, 169). It should be noted that young (~100g) rapidly growing animals were used in these previous 71

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72 studies and this may have contributed to the differences in the rates. However, our measured protein synthetic rates are similar to those measured in other adult rat skeletal muscles (e.g., 0.16%/h in quadriceps (16), 0.23%/h in gastrocnemius (28), and 0.33%/h in soleus (162)). Hence, the measured rates of MMP synthesis in the rat diaphragm in the current study are consistent with rates reported in the literature for adult animals. The MV-induced 30% decrease in MMP synthesis in the diaphragm occurred during the first 6 hours of MV. Additionally, the MV-induced decrement in protein synthesis remained steady after 12 and 18 hours of MV. This rapid change in skeletal muscle protein synthesis has also been observed during periods of inactivity in rat gastrocnemius. Indeed, 6 hours of hindlimb immobilization leads to a 37% decrease in MMP synthesis (28). Similarly, the observed decrease in MMP synthesis in immobilized rat gastrocnemius remains at this level after 2 days of immobilization (168). Therefore, the observed decrease in MMP synthesis after 12 and 18 hours of MV in the rat diaphragm is consistent with the rat hindlimb immobilization data (168) and indicates that the diaphragm, like other skeletal muscles, is sensitive to loading state. Once unloaded, via MV, protein synthesis in the diaphragm rapidly decreases and a new steady-state of protein synthesis is established. Myosin Heavy Chain Protein Synthesis MHC is an essential component of the contractile apparatus and constitutes ~25% of skeletal muscle mass (122, 182). Importantly, force generation is proportional to the amount of myofibrillar protein within the fiber and thus a decrease in the rate of MHC synthesis would lead to a decrease in the force generating ability of the diaphragm (17, 26, 125). Accordingly, the rate of MHC protein synthesis was measured after 6, 12, and 18 hours of MV and compared to time matched controls.

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73 These were the first experiments to measure the rate of MHC protein synthesis in the rat diaphragm. The rate of protein synthesis calculated using tissue fluid [ 13 C]leucine is ~0.1%/h in contracting diaphragm (SB 6). Previous investigations report MHC protein synthesis rates of ~0.1%/h in the rat quadriceps (16) and ~0.25%/h in the rat soleus (162). It is unclear why the rates in the diaphragm are not in line with those of the more active soleus. The observation that MHC protein synthesis rate is slower than the MMP synthesis rate suggests that MHC protein turns over slower (longer half-life) than other proteins in the MMP pool. The rates of MHC protein synthesis in the current study are ~60% less than the MMP synthesis rate. Previous studies report an ~40% difference in quadriceps (16) and an ~25% difference in soleus (162). The data in the current study suggest that the MHC protein pool in the diaphragm is turning over slower than MHC protein in other skeletal muscles. Six hours of MV resulted in an ~65% decrease in the rate of MHC protein synthesis. This decrease in MHC protein synthesis was maintained through 18 hours of MV. This rapid decrease in the rate of diaphragmatic MHC protein synthesis was more severe than the reported change after 5 hours of hindlimb unloading where a consistent but non-significant decrease was measured in the soleus (162). A possible explanation for the divergent findings is that during MV the diaphragm is not contracting but the soleus is free to contract (against little resistance) during hindlimb unloading. Hence, this level of activation in the soleus may serve to attenuate the decrease in protein synthesis during hindlimb unloading compared to MV. The change in MHC protein synthesis has not been measured during hindlimb immobilization but the synthetic rate of another essential

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74 contractile protein, -actin, has been examined (173). During the first 6 hours of hindlimb immobilization, -actin protein synthesis in the rat gastrocnemius decreases ~66% (173). The MHC protein synthesis data in the current study and the -actin protein synthesis data after 6 hours of hindlimb immobilization (173) indicate that skeletal muscle rapidly adapts to unloading by significantly decreasing the rate of contractile protein synthesis. Myosin Heavy Chain mRNA The fractional synthetic rate of specific proteins can be altered by pretranslational events leading to a decrease in the amount of a given mRNA (e.g., the rate of transcription or turnover of MHC mRNA). As discussed previously, MV significantly slows both MMP and MHC protein synthesis. Because MHC is the most predominant protein in skeletal muscle it was hypothesized that 6 to 18 hours of MV would alter pretranslational events in the diaphragm (i.e., MHC mRNA content would decrease). The data, however, do not support this hypothesis. As indicated in Figures 4-7 through 4-10 6 to 18 hours of MV did not alter MHC mRNA content in the diaphragm. These observations are consistent with previous studies of locomotor skeletal muscle disuse. In the rat soleus -MHC (slow) mRNA content does not significantly change after 5 hours or 7 days of hindlimb unloading, yet the synthetic rate of MHC protein decreases significantly (162). Further, 6 to 72 hours of hindlimb immobilization does not change the amount of -actin mRNA in the rat gastrocnemius but the synthetic rate of -actin protein was decreased ~65% during the first 6 hours (173). The data from the current study in conjunction with previous studies demonstrates that the rapid decrease in the rate of protein synthesis in the diaphragm, like other skeletal

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75 muscles, is not due to a change in MHC mRNA content. Nonetheless, a recent report indicates that extended periods of MV, >48 hours, does alter MHC mRNA (180). Using Northern blot analysis, this group reported that >48 hours of MV increases MHC IIa (70%) and MHC IIx (22%) mRNA, with little change in IIb MHC mRNA (4%), and no change in type I mRNA (180). Immunohistochemistry was used to fiber type a portion of diaphragm after >48 hours of MV and detected a significant decrease in type I fibers and a significant increase in fibers co-expressing both type I and II MHC protein (180). These findings (180), in conjunction with the present study, suggest that during the first 18 hours of MV there is no measurable change in MHC mRNA but over the course of the next 36 hours MHC mRNA expression does change and leads to a slow-to-fast MHC shift associated with skeletal muscle unloading. Regulation of Protein Synthesis Protein synthesis is the culmination of many events, including transcription and translation; all of which are highly regulated. The rapid decrease in protein synthesis after MV could be due to the inhibition of one or both of these steps. A discussion of key points of regulation of protein synthesis as they pertain to the MV-induced decrease in protein synthesis follows. Transcription In healthy active skeletal muscle, MHC protein expression appears to be regulated by transcriptional events (18). For example, -MHC promoter region activity in the soleus is significantly decreased after 7 days of inactivity (58, 80). Additionally, changes in mRNA expression precede changes in protein expression measured from the 4 th day to the 90 th day of inactivity (79). Thus, over a period of days/weeks/months MHC protein

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76 expression is regulated by transcriptional events but during the first hours of reduced use (e.g., 18 hours) protein expression is regulated by post-transcriptional events. MV did not change the amount of total RNA ( Table 4-5 ) or MHC mRNA ( Figures 4-7 through 4-10 ). Hence, the observed decrease in the rate of protein synthesis without a decrease in total RNA and MHC mRNA is indicative of a decrease in translational efficiency (the amount of MHC protein synthesized per amount of MHC mRNA). A decrease in translational efficiency occurs when one or more steps of translation is hindered. Translation Initiation The process of translating mRNA into a nascent polypeptide chain includes initiation, elongation, and termination. In the following sections control of initiation will be discussed in terms of relevant protein (initiation) factors and the pathway that controls the assembly of the initiation complex. This will be followed by a discussion focusing on the regulation of elongation and termination via the 3 end of mRNA. Translation initiation is the result of a series of steps culminating in the 40S and 60S ribosomal subunits binding to mRNA. The regulatory processes involved in initiation have been well elucidated. Specifically, proteins known as eukaryotic initiation factors (eIFs) are required for the engagement of the 40S ribosomal subunit and the 60S ribosomal subunit with mRNA. eIF function/activity is regulated by specific kinases and phosphatases. Of particular interest is the regulation of eIF4E by 4E-binding protein (BP)1. eIF4e is a critical component of the initiation complex and when bound by 4E-BP1 initiation is hindered. 4E-BP1 binding of eIF4E is regulated by Akt (also known as protein kinase B) and the putative kinase, mammalian target of rapammycin (mTOR)

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77 (144). Akt and mTOR are central components of the Akt/mTOR pathway, which is modulated by the loading state of skeletal muscle. Akt acts directly on mTOR and the activity of Akt is sensitive to disuse. After 2 weeks of hindlimb suspension Akt protein expression and phosphorylated Akt (active Akt) is significantly decreased (25). mTOR phosphorylation is modulated by Akt and 2 weeks of hindlimb suspension leads to a 60% decrease in mTOR phosphorylation (131). Further, 2 weeks of hindlimb unloading increases 4E-BP1 binding to eIF4E by >100% (25). The results of Bodine et al. (25) and Reynolds et al. (131) suggest that hindlimb unloading decreases the amount of Akt which in turn decreases mTOR activity leading to increased 4E-BP1 binding to eIF4E (25) and thus preventing eIF4E from participating in initiation. This is significant because it directly implicates the Akt/mTOR pathway in muscle atrophy by inhibiting initiation. In addition to controlling the phosphorylation state of 4E-BP1, the Akt/mTOR pathway controls the activity of the 70-kDa 40S ribosomal protein S6 kinase (p70 S6k ), possibly through mTOR and directly by protein dependent kinase 1 (PDK1) (144). After the phosphorylation of p70 S6k its activity increases. Control of p70 S6k is important because this kinase controls the function of the ribosomal protein S6, a component of the 40S ribosomal subunit that is involved in tRNA recognition (144). After 12 hours of hindlimb unloading or after 12 hours of denervation the phosphorylation state of p70 S6k is decreased ~ 3-fold and remains depressed after 7 days (76). The decreased phosphorylation state of p70 S6k has also been reported after 2 weeks of hindlimb suspension (25). Future experiments should explore the effect of MV on inhibition of translation initiation in the diaphragm.

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78 Translation Elongation and Termination In addition to impairing initiation, reduced use of skeletal muscle impairs elongation and termination. Previous studies (12, 88) indicate that during the initial hours of reduced use protein synthesis in skeletal muscle stalls during elongation. Polysome density is a measure of the number of ribosomes engaged in elongation per mRNA; increasing in the number of ribosomes per mRNA increases the density of polysomes. Ku and Thomason (88) studied -actin polysome density after 18 hours of hindlimb unloading and found a significant increase in polysome density. These data indicate that assembly of the ribosomes on mRNA, (i.e., initiation) continues during the first 18 hours of unloading. Further, increased polysome density indicates that elongation is somehow inhibited. As a follow up to the study of Ku and Thomason (88), Ashley and Russell (12) tested the hypothesis that the 3 UTR of the -MHC regulates the decrease in protein synthesis after 2 days of tenotomy in the rat soleus. Translation in the -MHC 3 UTR was significantly decreased (12). Importantly, they found a significant increase in the binding of a trans-acting protein factor in the 3 UTR (12). Based on these findings and the work of Ku and Thomason (88) the authors suggest the following model: During unloading the trans-acting protein binds to the -MHC mRNA 3 UTR with greater affinity (12). This binding would physically prevent the ribosomes from reaching the stop codon during elongation (i.e., translation would stall) (12). This would prevent a completed protein from being released (termination) and thus repress protein expression (12).

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79 In summary, the MV-induced decrease in MMP and MHC protein synthesis in the diaphragm may be due to alterations in the translational apparatus. Indeed, our finding that MV does not alter diaphragmatic MHC mRNA levels but results in a decreased rate of protein synthesis is consistent with this postulate. The current study did not measure rates of initiation or elongation, inhibition of the Akt/mTOR pathway and/or ribosomal stalling during elongation. However, one or more of these mechanisms may contribute to the rapid decrease in protein synthesis induced by MV. This is an interesting area for future research. Critique of the Experimental Model These experiments measured the changes in protein synthesis in the diaphragm during the initial hours of MV. Due to the invasive nature of these experiments the rat was used as the experimental model because of the biochemical and functional similarities between the rat and human diaphragm. The rate of incorporation of the stable tracer [ 13 C]leucine into diaphragmatic proteins was used to measure protein synthesis. To account for possible limitations of the experimental model a SB group was incorporated into the experimental design. The SB animals underwent a surgical procedure identical to the MV animals and received the same anesthetic for the same period of time. The SB animals, therefore, served as time matched controls for the MV group so that any alterations observed could be attributed to the effect MV. Of particular interest to these experiments are the following considerations: the animals were not fed, the possibility that anesthesia impacted protein synthesis, the length of the infusion period, and the removal of blood.

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80 Nutritional Status The use of 13 C[leucine] precluded feeding the MV and SB animals during the experiments. 13 C is a naturally occurring stable isotope present in all foods. Likewise, leucine is a branched chain amino acid present in protein sources. Thus, feeding the animals during the experiments would introduce an unknown amount of leucine with an unknown amount of 13 C and 12 C into the animal's circulation and tissue (Kevin Yarasheski, personal communication). This would dilute the 13 C[leucine] tracer administered to measure muscle protein synthesis by an unknown amount. Thus, the interpretation of the 13 C[leucine] enrichment data would be difficult if not impossible. Therefore, to avoid confounding the 13 C[leucine] enrichment measures, we chose not to feed the MV and SB animals during the experimental period. It was observed that protein synthesis decreased over time in the SB group. Comparing the SB6 group to the SB18 group the animals experienced a 32% decrease in the rate of MMP synthesis and a 41% decrease in the rate of MHC synthesis. The observed changes in the synthetic rate are consistent with the literature. Goldspink et al. (62) report a 48% decrease in diaphragm MMP synthesis 23 hours post feeding and Bates et al. (22) report a 44% decrease in the rate of limb-locomotor MHC synthesis 24 hours post feeding. However, comparing the MV18 results to the time matched SB group a 29% decrease in MMP synthesis and a 68% decrease in MHC synthesis is still realized. Therefore, the impact of MV on protein synthesis in the diaphragm was not obscured by the nutrient status of the animals. Anesthesia The anesthetic agent, sodium pentobarbital, could have impacted protein synthesis in the diaphragm. Nonetheless, rats anesthetized with 20 mg/kg sodium pentobarbital

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81 (twice the dose used in the current experiments) for 1 hour did not experience a significant decrease in protein synthesis in skeletal muscle (74). Additionally, general anesthesia does not decrease protein synthesis in skeletal muscle in healthy humans undergoing abdominal surgery (47). Collectively, these experiments indicate that protein synthesis is not altered by anesthesia per se. The influence of continued exposure of any given anesthetic agent (e.g., 18 hours) would be difficult to separate from the reduced use during that state. However, the experiments reviewed above (47, 74) report normal rates of protein synthesis in limb-locomotor skeletal muscle during periods of time that reduced use would not be expected to have an affect on protein synthesis. These reports (47, 74) indicate that anesthesia does not affect protein synthesis; therefore, the decreased rate of protein synthesis in the diaphragm during MV is attributable to MV, not the anesthetic. Infusion period Protein synthesis in the diaphragm was measured using the primed dose constant infusion method with the stable tracer [ 13 C]leucine. A plateau in 13 C enrichment of the plasma was achieved over the 6-hour infusion period ( Figure 4-1 ). It is unknown if such a plateau occurred within the tissue fluid of the diaphragm. The infusion period was 6 hours for all three experimental time points and tissue samples were taken at the completion of the experimental period. Early work by Fern and Garlick (50) demonstrated that a plateau in the enrichment of the plasma pool occurs within 2 hours but enrichment of the labeled free amino acid in the tissue fluid of the diaphragm continued to increase during 6 hours of infusion. The same authors (50) point out that if the free amino acid pool in the plasma or tissue reflects the labeled protein precursor then the calculated protein synthesis rate should be the same regardless of which precursor

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82 pool is used in the calculation. Despite the continued rise in tissue fluid enrichment, the authors (50) report similar rates of protein synthesis using each of the precursor pools, with the plasma precursor pool giving the slowest rates. Similar to Fern and Garlick (50), the rates of protein synthesis calculated using plasma [ 13 C]leucine in the present experiments underestimated the rates of protein synthesis. The concentration or enrichment of [ 13 C]leucine in the plasma should be greater than the enrichment of downstream precursor pools such as tissue fluid and the transamination product of leucine, KIC. Therefore, using plasma [ 13 C]leucine as a precursor pool to calculate protein synthesis should, and does, give a lower estimation of protein synthesis than tissue fluid or KIC enrichment. The data in the present experiments indicate that plasma [ 13 C]KIC and tissue fluid [ 13 C]leucine are appropriate surrogates of t-leucyl RNA. Indeed, using these surrogates to calculate rates of protein synthesis yields similar rates ( Table 4-4 ). The duration of the [ 13 C]leucine infusion period was 6 hours. This duration was chosen in order to achieve a plateau in [ 13 C]leucine enrichment of the plasma precursor pool (see Figure 4-1 ). Due to the length of the infusion period the rates of protein synthesis are a rolling average of the 6-hour period. Thus, the rates of protein synthesis at each time point may underestimate the actual rate of protein synthesis at any given moment. This would be the most profound during the first 6 hours of MV, as the last hour would be averaged in with the first. Blood Removal One milliliter of blood was drawn from each animal before [ 13 C]leucine infusion and after the 5 th and 6 th hours of infusion. Following each blood draw an equal volume of normal saline was administered to prevent hypovolemia. The initial blood sample and the

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83 sample taken after the 5 th hour precluded our ability to monitor arterial blood gas during the experiments. Nonetheless, we have demonstrated that our MV protocol results in only minor disturbances in blood gas homeostasis over a 24-hour period (125). However, SB animals typically experience some degree of respiratory acidosis without any significant affect on diaphragmatic contractile function (125). During the present experiments we relied on our previous experience with the mechanical ventilator and anesthesia parameters and it is possible that blood gas homeostasis was not adequately maintained. It should be noted that over the course of these experiments a high degree of surgical success was achieved (i.e., 88% of the experiments were successful) suggesting that animal homeostasis was well maintained despite our inability to monitor blood gas homeostasis. Mode of Mechanical Ventilation Pressure-assist MV is commonly used to treat adult patients in intensive care units. However, we used controlled MV for two reasons. First, because controlled MV results in rapid diaphragmatic atrophy (167) the impact of controlled MV can be studied during relatively short time periods. Second, controlled MV is clinically relevant as it is used in adult patients following drug over dose, spinal cord injury and is commonly used in certain pediatric situations (72). Summary and Future Experiments These experiments investigated the affect of MV on protein synthesis and MHC mRNA in the rat diaphragm. The hypothesis that MV-induced diaphragmatic atrophy is, at least in part, due to a decreased rate of total (MMP) and myofibrillar (MHC) protein synthesis was supported. However, the hypothesis that MV alters pretranslational events in the diaphragm was not supported.

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84 Future experiments investigating the mechanisms that regulate protein synthesis in the diaphragm during MV should follow several pathways. First, the role the Akt/mTOR pathway plays in regulating the synthesis of diaphragmatic proteins during MV should be studied. Secondly, polysome density of actin and myosin mRNAs after MV should be measured to determine if these mRNAs are acutely regulated by arresting translation during elongation. Additionally, identification of the trans-acting protein(s) binding to the MHC mRNA 3 UTR should be pursued.

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102 176. West JB and West JB. Pulmonary pathophysiology--the essentials. Baltimore, Md, Williams & Wilkins. 1998. 177. Witzmann FA, Kim DH, and Fitts RH. Hindlimb immobilization: length-tension and contractile properties of skeletal muscle. J Appl Physiol 53: 335-345, 1982. 178. Wright C, Haddad F, Qin AX, and Baldwin KM. Analysis of myosin heavy chain mRNA expression by RT-PCR. J Appl Physiol 83: 1389-1396, 1997. 179. Yang L, Bourdon J, Gottfried SB, Zin WA, and Petrof BJ. Regulation of myosin heavy chain gene expression after short-term diaphragm inactivation. Am J Physiol 274: L980-L989, 1998. 180. Yang L, Luo J, Bourdon J, Lin MC, Gottfried SB, and Petrof BJ. Controlled mechanical ventilation leads to remodeling of the rat diaphragm. Am J Respir Crit Care Med 166: 1135-1140, 2002. 181. Yarasheski KE, Smith K, Rennie MJ, and Bier DM. Measurement of muscle protein fractional synthetic rate by capillary gas chromatography/combustion isotope ratio mass spectrometry. Biol Mass Spectrom 21: 486-490, 1992. 182. Yates LD and Greaser ML. Quantitative determination of myosin and actin in rabbit skeletal muscle. J Mol Biol 168: 123-141, 1983. 183. Zhan WZ, Farkas GA, Schroeder MA, Gosselin LE, and Sieck GC. Regional adaptations of rabbit diaphragm muscle fibers to unilateral denervation. J Appl Physiol 79: 941-950, 1995. 184. Zhan WZ, Miyata H, Prakash YS, and Sieck GC. Metabolic and phenotypic adaptations of diaphragm muscle fibers with inactivation. J Appl Physiol 82: 1145-1153, 1997. 185. Zhan WZ and Sieck GC. Adaptations of diaphragm and medial gastrocnemius muscles to inactivity. J Appl Physiol 72: 1445-1453, 1992.

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BIOGRAPHICAL SKETCH R. Andrew Shanely earned a Bachelor of Science in Physical Education from California State University Fullerton in 1993. He then graduated from Northern Arizona University with his Master of Arts degree in Exercise Physiology in 1996. While at Northern Arizona University he investigated the effects of ammonia on the in vitro contractile properties of the rat diaphragm. His interest in diaphragm function led him to continue his graduate studies at the University of Florida, under the direction of Dr. Scott Powers. His studies at the University of Florida focused on the atrophic effects of mechanical ventilation on the diaphragm. Upon receiving his Ph.D. Andrew accepted a postdoctoral research position at the University of Missouri, under the direction Dr. Frank Booth. 103


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PROTEIN SYNTHESIS AND MYOSIN HEAVY CHAIN mRNA IN THE RAT
DIAPHRAGM DURING MECHANICAL VENTILATION
















By

R. ANDREW SHANELY


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2002




























Copyright 2002

by

R. Andrew Shanely




























The completion of this Dissertation is one my greatest accomplishments and is dedicated
to my parents. If it were not for your support and enthusiasm of my educational
development I would never have contemplated postgraduate work, let alone completed it.
Each of you has given me the love, understanding, encouragement, and freedom to lead a
happy and successful life. Thank you.

This Dissertation is also dedicated to Shannon. Your love, support, and daily
encouragement never ceases to amaze me. My love, thank you for your day-to-day help
and understanding throughout my postgraduate career.















ACKNOWLEDGMENTS

I would like to acknowledge those who made the successful completion of this

project possible: my mentor, Dr. Scott Powers; and my committee members, Dr. Stephen

Dodd, Dr. Randy Braith, and Dr. Paul Davenport. Collectively and individually, my

committee provided me with invaluable guidance, instruction, and patience. I would also

like to acknowledge Darin Van Gammeren, Michael McKenzie, and Murat Zergeroglu

for their contribution to this project, without which these experiments would not have

been possible. Kevin Yarasheski's measurement of [13C]leucine incorporation into

diaphragmatic proteins was the crux of these experiments. His enthusiastic participation

assured the success of these experiments. Fadia Hadad's knowledge and patience made

the measurement of myosin heavy chain mRNA a reality. Jeff Coombes, Haydar

Demirel, and Hisashi Natio must be acknowledged for their immediate guidance upon my

arrival in Dr. Scott Power's laboratory and thus their indirect contribution to this project.

I am forever indebted to Dr. Powers for serving as my mentor. His motivation,

guidance, support, mentorship, and friendship have been unflagging. A student is only

capable of what his mentor demands!

This work was made possible by funding from the National Institutes of Health

(HL-62361).
















TABLE OF CONTENTS
page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ............................................. .. .. .... .............. viii

LIST OF FIGURES ......... ........................................... ............ ix

A B ST R A C T .......... ..... ...................................................................................... x

CHAPTER

1 IN TR OD U CTION .............................................. .. ........... .............. .

Objectives of Specific Aim #1 .................. ...... ........................................... 2
Aim #1: Rationale for Experimental Approach and Hypothesis ................................... 2
Objectives of Specific A im #2 .................. ................. .............. ........ .............. 3
Aim #2: Rationale for Experimental Approach and Hypothesis ................................... 3


2 LITER A TU R E R EV IEW ................................................................. ....................... 4

H history of M mechanical V entilation........................................................................... 4
Indication for Clinical Use of Mechanical Ventilation........................................... 6
M odes of M echanical Ventilator Operation ........................................ .............. 8
Controlled M echanical V entilation................................................. ................. 8
A ssist-Control V entilation ........................... ....... .................................... 9
Intermittent M mandatory Ventilation ......................... ...................................... 9
Pressure Support Ventilation ................................... ....... .............. 10
Diaphragmatic Motion during Mechanical Ventilation............................................... 10
W meaning from M echanical V entilation............................................... .... .. .............. 12
Properties of the D iaphragm .......................................................... .............. 14
Function of the D iaphragm .................................... ................................................. 14
Metabolic Characteristics of the Diaphragm ......................................................... 15
Skeletal Muscle Fiber Types within the Diaphragm............................................ 16
O x idativ e C ap city ....................................................................... .................... 18
M uscle A trophy ...................................... ............ ......................... 19
M odels of Locom otor M uscle Atrophy ................................. .................................... 20
Reduced Electrical Activation and Load Bearing........................................... 20
Reduced Loading ........................................................... 22
M models of Inactivity .................. ............................ .. ...... .. .......... .. 24


v









M odels of Atrophy in the Diaphragm .................................... ................... ...... ...... 26
Procedure for Investigating Diaphragmatic Atrophy........................................... 27
Electrom yographic A activity ...................... .... ............ .................. .............. 27
Diaphragmatic Mass.......................... .. ... .. .................... 28
Differential Fiber Type Response to Diaphragmatic Inactivity.......................... 28
M yosin H eavy C hain C ontent...................................................... .... .. .............. 29
M yosin H eavy C hain m R N A ........................................................................ ... 29
C ontractile P roperties....................................................... ............. .......... 30
O xidative Capacity .................. ..................................... .. .......... .. 30
Protein Synthesis......................................... .......... 30
D iaphragm L ength C hanges................... .......................... .............. ... 31
Mechanical Ventilation and Diaphragmatic Atrophy ............................................. 32
E electrical A activity ............ .......... .................. ............ ... ............. .... ............. 32
D iaphragm atic D isuse A trophy..................................... ......................... .. ....... 32
Anim al M odels and M echanical Ventilation .................. .............................. ... 33
Effects of Mechanical Ventilation on Diaphragmatic Contractile Properties........ 34
Mechanical Ventilation and Diaphragmatic Atrophy ........................................ 35
Sum m ary of L literature R eview .......................................................................... ..... 37


3 M E T H O D S ........................................................... ................ 4 0

Experim ental D esign-Specific Aim #1 ........................................ ....... .............. 40
A nim als and Experim ental D esign............................................... ... ... .............. 40
M mechanical ventilation protocol ........................................... ............... 41
Postm ortem exam nation ................................................. ............... .... 43
Control animals (nonmechanically ventilated) protocol..............................43
M methods U sed: B iochem ical A ssays................................................ ... ................. 44
Tissue rem oval and storage............................................ ............... ... 44
Rates of in vivo diaphragmatic protein synthesis.....................................44
Statistical analy sis .................................................. ............ ...................... 49
Experim ental D esign-Specific A im #2 ................................... ................................... 49
M methods U sed: B iochem ical A ssays................................................ ... ................. 49
T otal R N A isolation ........... .............................................. ........ .............. 49
R everse transcription (R T)........................................ ........................... 50
Polymerase chain reaction (PCR) ................................................ 51
A analysis of gels ............. ......... ................ ...............................................52
Statistical analysis ....... ................................ .. .. .... ...... .. ........ .... 53


4 R E S U L T S ................................................................................................................. 5 4

Morphological, Physiological, and Post Mortem Observations ................................. 54
Influence of Mechanical Ventilation on Protein Synthesis......................................... 55
Total RNA and Myosin Heavy Chain mRNA in the Diaphragm after Spontaneous
Breathing and M mechanical Ventilation .......................................... ..... ......... 57









5 D IS C U S S IO N ........................................................................................................... 7 1

O verview of Principle Findings ............................................................... ............... ... 71
Impact of Mechanical Ventilation on Protein Synthesis in the Diaphragm .............. 71
M ixed M uscle Protein Synthesis.................................................. .................... 71
M yosin H eavy Chain Protein Synthesis............................................. ... ................. 72
M yosin H eavy C hain m R N A ........................................................................ ... 74
R regulation of P rotein Synthesis.......................................................... .... ................ 75
Transcription ..... ................................ ................... 75
Translation Initiation.................................. .......................... ......76
Translation Elongation and Termination ............................... ................ 78
Critique of the Experim ental M odel ..................................... ........................ .......... 79
Nutritional Status ......................................... .............. 80
A anesthesia ..................................................................................................... 80
Infusion period ............................................... ............. .......... 81
Blood Removal ......................... ......... .............. 82
M ode of M echanical V entilation ........................................ ....................... 83
Sum m ary and Future Experim ents...................................................... .... .. .............. 83


L IST O F R E FE R E N C E S ....................................................................... ... ...................85

BIOGRAPHICAL SKETCH ............................................................. ............... 103
















LIST OF TABLES


Table page

2-1 Fiber type composition (%) of the diaphragm and locomotor skeletal muscles. ........17

2-2 Bioenergetic enzyme activities in the costal diaphragm and two locomotor
m u sc le s ...................................................................... 1 9

3-1 Oligonucleotide primers used for the PCR reactions ...............................................53

4-1 Animal body mass before and after experimental period.........................................58

4-2 Heart rate response during M V and SB.................................................................... 59

4-3 Systolic blood pressure response during MV and SB ............................................59

4-4 Fractional synthetic rates of mixed muscle protein and myosin heavy chain
protein by calculation with each surrogate of the [13C]leucyl-tRNA precursor
p o o l. ........................................ .................... ................ 6 5

4-5 Total RNA obtained from the costal diaphragm ............................... ............... .66















LIST OF FIGURES


Figure p

3-1 Experim mental design for Specific Aim #1. ........................................ .....................41

3-2 Schematic representation of the myosin heavy chain (MHC) genes...........................53

4-1 Plasma [13C]leucine and plasma [13C]ketoisocaproic acid ([13C]KIC) enrichment. ...60

4-2 Tissue fluid [13C]leucine enrichment in the diaphragm ............... .................. 61

4-3 Mixed muscle protein and myosin heavy chain [13C]leucine enrichment in the
diaphragm ....................................................... ................. 62

4-4 Fractional synthetic rates of mixed muscle protein (MMP) by calculation with
tissue fluid [13C leucinee. ............................................................. .....................63

4-5 Fractional synthetic rates of myosin heavy chain (MHC) protein by calculation
w ith tissue fluid [13C leucinee. ........................................ .......................... 64

4-6 R T-PCR products. ......................... ........................ .. ............. ......... 67

4-7 Relative type I M HC expression. ........................................ ........................... 67

4-8 Relative type IIa M H C expression. ........................................ ......................... 68

4-9 Relative type IIx M HC expression. ........................................................................... 69

4-10 Relative type IIb M HC expression. ........................................ ....................... 70















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

PROTEIN SYNTHESIS AND MYOSIN HEAVY CHAIN mRNA IN THE RAT
DIAPHRAGM DURING MECHANICAL VENTILATION

By

R. Andrew Shanely

December 2002
Chair: Scotty K. Powers, Ph.D., Ed.D.
Department: Exercise and Sport Sciences

The purpose of these experiments was to test the hypothesis that mechanical

ventilation-induced diaphragmatic atrophy is due, at least in part, to a decreased rate of

total protein synthesis and myosin heavy chain (MHC) protein synthesis. We also tested

the hypothesis that mechanical ventilation (MV) alters pretranslational events in the

diaphragm. To test these hypotheses, we randomly assigned specific-pathogen-free

barrier-protected 4-month-old female Sprague-Dawley rats to one of three experimental

groups: MV; spontaneously breathing (SB); or control/acute anesthesia. The MV animals

were mechanically ventilated for 6, 12, or 18 hours (n=10 for each time period).

Spontaneously breathing animals underwent the same surgical procedures and were

anesthetized for the same period of time as the animals in the MV group, but were not

exposed to MV. The acute-control animals (n=10) were not exposed to MV or prolonged

anesthesia. The rate of protein synthesis was determined by measuring the rate of

[13C]leucine incorporation into total protein and MHC protein in the diaphragm of the









MV and SB rats. We isolated total RNA from the diaphragms of all groups and measured

the expression of type I, IIa, IIx, and IIb (the four adult MHC mRNA isoforms). The rate

of protein synthesis of the MV rats was compared to that of the SB rats at the 6, 12, and

18 hour time points. Six hours of MV caused a significant decrease (30%, p < 0.05) in the

rate of total protein synthesis and a significant decrease (65%, p < 0.05) in the rate of

MHC protein synthesis. The decrease (p < 0.05) in protein synthesis remained at this

depressed level after 12 and 18 hours of MV. Expression of MHC mRNA isoforms in the

diaphragms of MV animals and the SB animals did not change (p > 0.05). These data

support the hypothesis that a decrease in protein synthesis contributes to MV-induced

diaphragmatic atrophy. In contrast, these data do not support the hypothesis that MV

alters pretranslational events in the diaphragm.














CHAPTER 1
INTRODUCTION

Mechanical ventilation (MV) provides a means of supporting blood gas

homeostasis for patients who cannot maintain adequate alveolar ventilation.

Unfortunately, prolonged MV (i.e., > 3 days) is not without consequence because as

many as 20% of patients experience difficulty in "weaning" from the ventilator (94).

While the underlying cause for weaning difficulties has yet to be fully elucidated,

respiratory muscle atrophy and the associated contractile dysfunction are potential

mechanisms (167).

In this regard, Anzueto et al. (8) reported significant reductions in diaphragmatic

strength and endurance of healthy baboons after 11 days of MV. Despite these important

physiological findings, Anzueto and colleagues' report did not investigate biochemical or

histological alterations in the diaphragm associated with MV. Further, Le Bourdelles et

al. (92) examined the effects of 48 hours of controlled MV on both atrophy and

contractile properties in the rat diaphragm. They reported a significant reduction in

isometric force generation and a reduction in both diaphragmatic mass (i.e., atrophy) and

protein content (92). Powers et al. (125) have also reported that MV leads to progressive

diaphragmatic contractile dysfunction. These experiments demonstrated a significant

correlation between time on the ventilator and contractile dysfunction (i.e., the greater the

time on the ventilator the greater the degree of diaphragmatic contractile dysfunction)

(125). Finally, recent experiments in our laboratory have demonstrated that MV for little

as 18 hours results in diaphragmatic atrophy (145). Preliminary experiments in our









laboratory suggest that the observed diaphragmatic atrophy is associated with a decreased

rate of diaphragmatic protein synthesis and a decrease in myosin heavy chain (MHC)

content. These observations form the basis for the proposed experiments.

Objectives of Specific Aim #1

The effect of MV on diaphragmatic protein synthesis was determined. We tested

the hypothesis that MV-induced diaphragmatic atrophy is due, at least in part, to a

decreased rate of total and myofibrillar protein synthesis.

Aim #1: Rationale for Experimental Approach and Hypothesis

Preliminary experiments in our laboratory indicated that prolonged MV results in

significant diaphragmatic atrophy. The extent to which decreases in protein synthesis

contribute to the MV-induced loss of diaphragmatic contractile protein is unknown.

Therefore, these experiments were designed to determine the time course of changes in

protein synthesis during 6, 12, and 18 hours of MV. This was achieved by measuring

both total and contractile protein synthesis rates (in vivo) in the diaphragms of control and

MV animals. Specifically, diaphragmatic protein synthesis was measured over the course

of the last 6 hours of the experimental period (e.g., 12 tol8 hours of MV). The fractional

rate of diaphragm muscle protein synthesis was measured using intravenous infusion of

[1-13C]leucine. We quantified the in vivo rate of incorporation of [1-13C]leucine into both

total and contractile proteins in the diaphragm by gas chromatography-combustion-

isotope ratio mass spectrometry (GC-C-IRMS). The stable isotope [1-13C]leucine was

chosen for several reasons:

* Less isotope effect (i.e., less tissue injury).
* Small tissue sample is required for analysis.
* Safety.









* Validity and reliability of this label for measurement of the rate of in vivo muscle
protein synthesis is well established (121).

Objectives of Specific Aim #2

The effect of MV on diaphragmatic myosin heavy chain (MHC) mRNA content

was determined. We tested the hypothesis that MV alters pretranslational events in the

diaphragm.

Aim #2: Rationale for Experimental Approach and Hypothesis

Preliminary experiments suggested that prolonged MV results in a decrease in

diaphragmatic protein synthesis. The extent to which pretranslational events contribute to

the MV-induced decrease in protein synthesis is unknown. Therefore, these experiments

were designed to determine the time course of changes in MHC mRNA after 6, 12, and

18 hours of MV. This was achieved by measuring the MHC mRNA content in

diaphragms from control and MV animals. Specifically, we measured the diaphragmatic

content of type I, IIa, IId/x, and IIb MHC mRNA in control animals and at the

completion of 6, 12, and 18 hours of MV.














CHAPTER 2
LITERATURE REVIEW

Mechanical ventilation (MV) is an intervention used to sustain ventilation in

patients who are unable to maintain adequate alveolar ventilation. The withdrawal of MV

is commonly referred to as "weaning." Patients who experience weaning difficulties

commonly exhibit respiratory muscle weakness. Hence, it has been postulated that both

weakness and decreased endurance of respiratory muscles are major contributors to the

failure to wean patients from MV (167). This notion is strongly supported by recent

animal studies indicating that prolonged controlled MV results in significant reductions

in diaphragmatic force production. Further, our laboratory recently discovered that the

MV-induced diaphragmatic force deficit is associated with significant diaphragmatic.

History of Mechanical Ventilation

Galen (56), in the year AD 160, may have been the first to artificially ventilate an

animal. He reported that "If you take a dead animal and blow air through its larynx

(through a reed), you will fill its bronchi and watch its lungs attain the greatest

distention." More than 1000 years after Galen, Vesalius found that he could keep the

heart beating after a pneumothorax by inflating the lungs through a reed tied to the

trachea (171). In 1664 Hooke described dissecting a dog, putting a pipe into the trachea

and attaching the pipe to a bellows (15). The heart continued beating and the dog stayed

alive for over an hour (15).

Artificial ventilation of dogs led to the use of positive pressure ventilation to revive

human drowning victims in the mid 1700s (35). However, positive pressure ventilation









frequently caused fatal pneumothoraxes during animal experiments and was later

condemned by both the Academie Francaise and the Royal Humane Society (35).

Quashing positive pressure ventilation early in its development led to an alternative

method: negative pressure ventilation. Development and use of negative pressure

ventilation flourished in the 1800s and by 1928 the "Iron Lung" became the first negative

pressure ventilator used successfully on a large scale (35). The iron lung saved many

lives during the poliomyelitis epidemic in the 1930s and served as the mainstay of

treatment for respiratory paralysis from poliomyelitis until positive pressure ventilation

was reintroduced in the 1950s (35).

Positive pressure ventilation was heavily used in physiology laboratories during the

mid to late 1800s and by 1879 the volume-cycled ventilator was a common piece of

equipment at Harvard University (35). While positive pressure ventilation was used to

some degree before the 1950s, it was not until the poliomyelitis epidemic struck

Copenhagen in 1952 that its full utility was realized (35). Since then, positive pressure

ventilation has been used successfully to treat many medical conditions that lead to

respiratory insufficiency.

In the 1980s a new method of positive pressure ventilation was introduced. This

new application was a noninvasive means of ventilation via a nasal, facemask, or an oral

connection and proved to be a significant development in MV (133). The evolution of the

mechanical ventilator has continued to this day. Its use as an indispensable life-saving

tool has insured its place in clinical practice.









Indication for Clinical Use of Mechanical Ventilation

MV is used for 4 main reasons:

Life support for a patient with a life threatening illness whose recovery is
anticipated.
Life support for a patient under general anesthesia during surgery.
To provide ventilation during respiratory muscle failure or to compensate for a
damaged upper airway.
As an aid during recovery or rehabilitation from an illness (3).

A common physiological outcome of these situations is respiratory failure. Respiratory

failure is often defined as a PaO2 of less than 50 mmHg at sea level while breathing a gas

mixture of at least 50% 02 and/or a PaCO2 greater than 50 mmHg hypercapniaa) (3).

Respiratory failure due to inadequate gas exchange is termed hypoxic respiratory

failure (134). If respiratory failure is due to ventilatory pump failure it is known as

hypercapnic respiratory failure, or the two may occur in combination (134). Hypoxic

respiratory failure is commonly associated with severe respiratory illnesses and can

induce hypoxemia by one or some combination of four mechanisms: alveolar

hypoventilation, right-to-left shunt in the heart, ventilation-perfusion mismatch, or

incomplete diffusion equilibrium (176).

The rib cage and its muscles, the diaphragm, and the abdomen and its muscles

make-up what is known as the ventilatory pump (160). Alveolar ventilation and gas

exchange depend on the ventilatory pump. Hypercapnia is a telltale sign of ventilatory

pump failure. A reduction in central neural drive, inspiratory muscle impairment, and/or

excessive respiratory workload can induce ventilatory pump failure (3). Neuromuscular

disease, drug overdose, or brainstem injury can impair central drive to the point of

inducing ventilatory pump failure (3). Inspiratory muscle performance can be negatively

impacted by neuromuscular disease (4), metabolic disturbances (3), certain drugs (2), a









disadvantageous length-tension relationship (175), mechanical disadvantage (103),

altered force-velocity relationship (3), detraining and atrophy (8, 92, 125), and fatigue (2,

135). Excessive inspiratory muscle workload due to obesity, asthma, or pulmonary

resection for example, can also lead to respiratory pump failure.

Increased inspiratory muscle workload results in an increased work of breathing.

An increased work of breathing requires recruitment of the diaphragm (the primary

muscle of inspiration) and also recruitment of the accessory inspiratory muscles. This

presents two significant challenges: increased workload for the diaphragm and increased

oxygen consumption. The diaphragm is well suited to the constant demand of pulmonary

ventilation. However, if the constant workload exceeds 40% of its maximal force-

generating ability, it will fatigue (135) and ventilatory pump failure may ensue.

A further consideration of an increased work of breathing is the increased demand

for oxygen by the respiratory muscles. The respiratory muscles may account for more

than 50% of the total body oxygen consumption, as compared to less than 5% under

normal conditions (13). The increased oxygen cost of breathing reduces the availability

of oxygen to other body tissues and may lead to other potentially fatal events (e.g.,

myocardial ischemia) (167). As mentioned above, an increased work of breathing, if

excessive, may lead to diaphragmatic fatigue and thus hypoventilation. Hypoventilation

causes a loss of oxygen intake and also causes hypercapnia (which significantly impairs

muscle contractility) (85). This series of events, if left unchecked, can put the patient into

a downward spiral of respiratory muscle distress, hypoventilation, and hypercapnia (167).

This scenario may be remedied, however, by placing the patient on MV and thus









"resting" the diaphragm. In fact, mask ventilation typically improves respiratory

frequency, arterial oxygen tension, and pH soon after it is applied (6, 31).

Modes of Mechanical Ventilator Operation

The mode of MV depends on the needs of the patient (central drive, respiratory

muscle dysfunction, etc.). Controlled MV, assisted-control ventilation, intermittent

mandatory ventilation, and pressure support ventilation are all modes of MV commonly

used to aid the patient. The properties of each are outlined below.

Controlled Mechanical Ventilation

Controlled MV (CMV) is perhaps the most straightforward mode of MV.

Controlled MV delivers all breaths in a predetermined fashion. Breathing frequency (f),

tidal volume (VT), inspiratory-to-expiratory timing (I:E ratio), and inspiratory flow

pattern are each regulated by the ventilator settings. The patient's breathing frequency or

inspiratory effort cannot alter the preset respiratory parameters; hence, patient triggering

is not possible during CMV. Controlled MV is achieved pharmacologically (e.g.,

sedation and neuromuscular blockade) or by mechanical hyperventilation (78). The use of

CMV is limited to patients who are apneic because of brain damage, sedation, or

neuromuscular blocking agents (167). Controlled MV is used to treat hypoxemic

respiratory failure due to widespread atelectasis, localized alveolar disease,

noncardiogenic pulmonary edema, and cardiogenic pulmonary edema (3). Controlled MV

is also used to treat hypercapnic respiratory failure due to acute neuromuscular disease

and acute obstructive disease (3).

While CMV provides the maximum degree of respiratory muscle rest it is not

without consequence to the very muscle it is intended to aid, the diaphragm.

Administration of CMV often requires the use neuromuscular blocking agents whose use









is associated with prolonged weakness or paralysis lasting up to 1 week (68, 89, 143).

The effect of the neuromuscular blocking agents is compounded when corticosteroids are

given. The combination of neuromuscular blocking agents and large doses of

corticosteroids can result in generalized myopathy lasting weeks or months (44). Further,

this pharmacologic combination is associated with a higher incidence of muscle

weakness (93). Even in the absence of disease, CMV can induce muscle weakness which

is attributed to muscle atrophy (167).

Assist-Control Ventilation

Assist-control (AC) ventilation is the first mode of MV used in many institutions

(104). Assist-control ventilation provides a positive pressure breath in response to the

patient's inspiratory effort. The VT of each breath is set at the ventilator. The VT is

delivered with each inspiratory effort -or if the patient fails to trigger the ventilator

within a set amount of time. Assist-control allows the patient to control breathing

frequency. The patient also controls the ventilator-generated pressure. As the inspiratory

effort generated by the patient increases, the ventilator-generated assistance decreases

(104). Under the most favorable conditions, AC is 50-66% more effective than active

chest inflation at reducing respiratory work of breathing by (108).

Intermittent Mandatory Ventilation

Intermittent mandatory ventilation (IMV) provides a preset number of positive

pressure breaths and allows the patient to breathe spontaneously between ventilator-

delivered breaths. Intermittent mandatory ventilation can be set to provide a breath that is

either a preset volume or pressure. Once a predetermined pressure is reached, the

ventilator terminates the positive pressure breath. Further, IMV allows the patient to

autonomously alter his/her spontaneous breathing pattern. Because each IMV breath is









synchronized with the patient's breathing pattern, this mode of ventilation is also known

as synchronized intermittent mandatory ventilation (SIMV).

While IMV gives the clinician great flexibility in treating the patient, it has a

potential drawback. Intermittent mandatory ventilation was designed to provide volume

assistance while allowing the patient to breathe spontaneously (that is, to rest the

inspiratory muscles and attenuate inspiratory muscle deconditioning). However, when

IMV accounts for 20 to 50% of the total ventilation, the electromyographic (EMG)

activity of the diaphragm and the sternomastoid muscles is equal to that of spontaneous

breaths (81). While IMV is intended to provide inspiratory muscle rest, the EMG data

suggest that this may not be the case.

Pressure Support Ventilation

Pressure support ventilation (PSV) is designed to augment the patient's inspiratory

effort by providing positive pressure support. Pressure support ventilation reduces the

work of breathing by raising the airway pressure to a predetermined level after the patient

initiates a breath; and continues to do so until the end of the inspiratory effort is sensed as

a reduction in inspiratory flow (31). In PSV treatment, breathing frequency, VT, and

inspiratory flow pattern are determined by the patient. Pressure support ventilation is

widely used in intensive care units because it does not require heavy sedation of the

patient and it allows the patient to breathe spontaneously. Further, the patient is required

to use their inspiratory muscles, thereby reducing the severity of inspiratory muscle

deconditioning.

Diaphragmatic Motion during Mechanical Ventilation

The diaphragm, like the lung, can be described in terms of its position relative to

the pressures acting on it. In the upright position there is a vertical gradient in pleural









pressure acting on the lungs, and the pressure acting on the upper portion of the lungs is

more subatmospheric than the pressure at the bases of the lungs. The region of the lungs

at the bottom of the vertical gradient is termed dependent and the region at the top of the

gradient is termed nondependent. Therefore, while in the upright position the bases of the

lungs are in the dependent region, and the apices are in the nondependent region. Shifting

the body position to the supine position changes the dependent and nondependent

relationships (i.e., the dorsal region of the lungs is in the dependent region and the ventral

surface of the lungs is in the nondependent region).

Likewise, while in a supine position, the ventral portion of the costal diaphragm is

in the nondependent position and the dorsal portion of the costal diaphragm and the crural

portion of the diaphragm are in the dependent region. The middle costal portion of the

diaphragm lies between both regions but is often considered to be dependent. While

breathing spontaneously in the supine position, the dependent diaphragm is displaced or

has a greater excursion than the nondependent diaphragm (54, 151, 87) because of

anatomical differences between the costal and crural diaphragm region (87).

After anesthesia and MV, the position of the diaphragm at functional reserve

capacity (FRC) shifts cephalad (23, 54, 87, 129). The cephalad shift is due to loss of

muscle tone in the diaphragm and gravitational displacement of the abdominal contents

(23, 54, 129). After CMV the pattern of displacement is reversed: the dependent regions

of the diaphragm are displaced less than the nondependent regions of the diaphragm (54).

This reversal of displacement is the result of a uniform increase in thoracic pressure

displacing the diaphragm where abdominal pressure is least (i.e., the nondependent









region of the diaphragm) (54). Thus, during MV the diaphragm is passively moved each

time a breath is artificially delivered to the patient.

The diaphragm does not shorten to the same extent during MV as during

spontaneous breathing. The diaphragm does, however, shorten passively while being

displaced by the artificially ventilated lungs (119). The degree to which the diaphragm

passively shortens during MV is not uniform. Diaphragmatic shortening during PSV (87,

129) and CMV (151) has been reported to be less than that of spontaneous breathing.

However, others (53, 132) have reported greater diaphragmatic shortening during CMV

than during spontaneous breathing (119).

The disparity between these findings may be due to differences in methodology.

The studies that reported less diaphragmatic shortening during MV used indirect methods

such as 3-dimensional x-ray tomography (87), CT scans (129), and videofluoroscopy

(151) to measure diaphragmatic length. The studies that reported greater diaphragmatic

shortening during MV used Sonomicrometry (119). Sonomicrometry is a more accurate

and perhaps more reliable method for measuring muscle length changes. Despite the

different length changes reported during MV, the unifying finding is that the diaphragm

shortens passively. In addition to passive shortening, the diaphragm is also displaced by

the lungs during MV.

Weaning from Mechanical Ventilation

The common term for discontinuation of MV is "weaning." This term refers to the

slow withdrawal of MV at a rate the patient can tolerate. The weaning success rate in

many intensive care units (ICU) is usually higher than 70% depending on the subset of

patients (94). A person is considered a "weaner" if he/she is breathing spontaneously 2









days after discontinuation of MV (94). A patient who requires some degree of ventilatory

support (total or partial) is considered a "non-weaner" (94).

It is imperative to discontinue use of MV as soon as possible because MV is

associated with several major complications. Most patients requiring short-term MV

experience little difficulty when MV is withdrawn. As previously discussed, MV is

frequently used to aid patients recovering from respiratory failure. Discontinuing MV for

many of these patients is difficult. Because of the factors that led to the patients'

placement on MV, great care is given to their discontinuation from MV. The process of

discontinuation from MV is challenging and makes up a large portion of the ICU

workload (166).

The process of weaning may require more than 2 days and it can become a lengthy

process. "A long-term ventilator-assisted individual is a person who requires mechanical

ventilatory assistance for more than 6 hours a day for more than 3 weeks after all acute

illnesses have been maximally treated and in whom multiple weaning attempts by an

experienced respiratory care team have been made" (105). While MV may not be the

primary reason a patient is in the hospital, it is often the reason for a prolonged stay. The

total number of difficult weaners ranges from 20% up to 70% in some ICUs (94) and the

cost to treat these patients is large (105). A 1983 study estimated that there were 6,800

long-term MV patients at a cost of $1.7 billion per year or -1.5% of total hospital costs

(106). In 1990, the American Association for Respiratory Care commissioned a rigorous

study of the incidence and cost of long-term MV care (7). This study found that there

were 11,419 long-term MV patients at an annual cost of $3.2 billion for treatment (7).









While these studies relay the magnitude and dollar cost of MV, the importance of the loss

of individual independence as well as the emotional cost should not be forgotten.

Properties of the Diaphragm

The diaphragm is the primary muscle of inspiration. As such, it is chronically

active and its metabolic characteristics reflect this. This section reviews the functional

and metabolic characteristics of the diaphragm.

Function of the Diaphragm

Breathing has long been recognized as a vital process. For example, around 2000

BC, Chinese philosophers wrote about "lien ch'i," the process of bringing the inspired

breath into the soul substance (35). The ancient Greeks also believed that breathing was

essential and that the diaphragm was the seat of the soul (102). The Greek word phrenes

means soul; this is how the phrenic nerves were named (102). In the third century BC, the

diaphragm was recognized to be a muscle by Erasistratus and he taught that it was the

main muscle of inspiration (43).

The diaphragm is chronically active skeletal muscle and is innervated by the

phrenic nerves from the cervical segments 3, 4, and 5 (176). The diaphragm has two

functionally different and distinct parts: the central tendon (the non contractile portion)

and the costal and crural regions (the muscular portions). The costal and crural muscle

fibers extend outward from the central tendon. The fibers of the crural diaphragm radiate

from the central tendon and insert onto the anterolateral aspect of the first three lumbar

vertebrae and the aponeurotic arcuate ligaments (39). The fibers of the costal diaphragm

extend from the central tendon and insert on the xyphoid process of the sternum (the

ventral region) and the upper margins of the lower 6 ribs (the medial and dorsal regions)









(39). The muscle fibers of the costal diaphragm run cranially from their insertions and are

thus apposed directly to the inner aspect of the lower rib cage (39).

Chest wall displacement during inspiration is accomplished by the unique shape

and location of the diaphragm. The healthy diaphragm is an elliptical cylinder capped by

a dome (39). The dome region is primarily composed of the central tendon. The

cylindrical region is the portion apposed directly to the inner aspect of the lower rib cage,

the "zone of apposition" (39). This shape and location gives the diaphragm the ability to

increase chest wall dimensions and therefore inflate the lungs (123).

Activation of the diaphragm elicits a caudal force onto the central tendon and a

cephalic force onto the lower 6 ribs by the costal diaphragm and vertebral column by the

crural diaphragm (123). The caudal force causes the dome of the diaphragm to descend

and displace the abdominal contents downward, but the abdominal contents resist

displacement and therefore act as a fulcrum (160). Thus, as the diaphragm contracts and

its fibers shorten, the transverse dimensions of the chest wall increase (123, 160).

Contraction of the diaphragm also increases the cephalo-caudal dimensions of the chest

wall (123). Based on the insertions of the costal and crural diaphragm, the chest wall

dimensions are changed by the costal region as the crural region inserts onto the

immovable vertebral column and only displaces the abdomen (123, 160).

Metabolic Characteristics of the Diaphragm

The anatomical and morphological design of the diaphragm is well suited to the

constant demand of the ventilatory system. Likewise, the metabolic design of the

diaphragm allows it to meet the constant challenge imposed on it.









Skeletal Muscle Fiber Types within the Diaphragm

Skeletal muscle is a highly plastic tissue (it has the ability to adapt to the workload

imposed on it). Skeletal muscle meets the workload by expressing fiber types best suited

to the demand. Thus, the heterogeneity of skeletal muscle fibers expressed within a

muscle is a reflection of the "job" the muscle is responsible for. The diaphragm is a

highly specialized skeletal muscle. It is the only skeletal muscle that is chronically active

for life and its fiber type expression reflects this.

Fiber typing has evolved a great deal beyond the initial classification of "red" and

"white" put forth by Ranvier in 1873 (128). As reviewed by Pette and Staron (120)

muscle fibers can be classified by various methods including histochemistry,

immunohistochemistry, and gel electrophoresis.

Histochemical classification is a subjective method based on myofibrillar

actomyosin adenosine triphosphatase (ATPase) activity or aerobic and anaerobic

metabolic enzymes. These methods typically reveal 3 fiber types: I, IIa, and IIb (via

ATPase) or slow-twitch oxidative, fast-twitch oxidative glycolytic, and fast-twitch

glycolytic (via enzymatic analysis) (120). Immunohistochemistry and gel electrophoresis

are able to resolve 4 fiber types based on myosin heavy chain (MHC) proteins: I, IIa,

IId/x, and IIb. Immunohistochemistry and gel electrophoresis have been used to

determine the fiber type composition of rat and human diaphragm (Table 2-1). Overall

however, the human and the rat diaphragm are very similar in fiber type composition

(49).

Recently, the MHC content of human single muscle fibers were thoroughly

characterized histologically, immunohistologically, and electrophoretically (46, 140).

This information was then correlated with the mRNA transcripts for each MHC gene.









Table 2-1. Fiber type composition (%) of the diaphragm and locomotor skeletal muscles.


Muscle
Rat DIA
COD
COD
COD
COD
DIA
COD
COD
COD
COD
PL
PL
PL
PL
SOL
SOL
SOL
Human DIA
COD
COD
COD
COD
COD
SOL
SOL
VL
VL


Type
IId/x IIb
20
32
40
18 32
26
53 1
38 3
41 12
30 5
43 10
41
46 51
40 43
50 28
0
0 0
0 0
25
24
23
17
17
18
0
0
12
4


Method
HC
HC
HC
HC
HC
SDS-PAGE
SDS-PAGE
SDS-PAGE
IH
IH
HC
SDS-PAGE
SDS-PAGE
SDS-PAGE
HC
SDS-PAGE
SDS-PAGE
HC
HC
HC
SDS-PAGE
SDS-PAGE
IH
HC
SDS-PAGE
HC
SDS-PAGE


All values reported are percentages. Because of rounding, values may not total 100%.
DIA = whole diaphragm. COD = costal diaphragm. PL, plantaris; SOL, soleus; VL,
vastus lateralis; HC, histochemistry; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide
gel electrophoresis; IH, immunohistochemistry.


The mRNA transcripts in all fibers classified as lib using conventional methods are

actually equivalent to the rat IIx gene (46, 140). Based on these findings it was suggested

that all type II fibers previously identified as lib would be more accurately classified as


Reference
49
111
63
41
124
19
156
124
90
145
11
19
157
124
11
157
19
49
100
114
163
97
97
69
69
69
69









IIx (46, 140). Additionally, conventional histochemical techniques may have erroneously

classified rat IIx fibers as lib (120). In light of these findings, the diaphragms from the rat

and human are quite similar, as neither has many type lib fibers.

Oxidative Capacity

The oxidative capacity of skeletal muscle is consistent with its function. Skeletal

muscles that contract tonically are slow twitch and highly oxidative, whereas muscles

used sporadically are fast twitch and have a low oxidative capacity. The characteristics of

the diaphragm fit this biochemical tenet. Most muscle fibers in the diaphragm are highly

oxidative and this is in-line with the predominant fiber types in the diaphragm, type I and

IIa.

Typical biochemical markers of skeletal muscle metabolic pathways include citrate

synthase (CS), succinate dehydrogenase (SDH), 3-hydroxyacyl-CoA dehydrogenase

(HADH), hexokinase (HK), phosphofructokinase (PFK), and lactate dehydrogenase

(LDH). The oxidative capacity of a muscle is often estimated by the citric acid cycle

enzymes CS and SDH. Likewise, the lipolytic capacity can be determined by an integral

P-oxidation enzyme, HADH. The enzymes HK, PFK and LDH are often used to

determine the glycolytic capacity of muscle cells

To date, there are two published reports of normal human diaphragmatic

bioenergetic enzyme capacities (139, 163). Sanchez et al. (139) compared the

bioenergetic capacity of the diaphragm to the latissimus dorsi. In each case,

thebioenergetic capacity of the diaphragm was significantly greater than that of the

latissimus dorsi (i.e., CS 180%, HADH 215%, HK 170%, and LDH 115%).









Table 2-2 shows the bioenergetic similarities of the human and rat costal

diaphragm, as well as two different locomotor muscles. Note that the bioenergetic

capacity of the human and rat diaphragm are very similar. Overall, the bioenergetic

capacity of the diaphragm reflects its continuous use, a high oxidative capacity as

measured by citric acid cycle and P-oxidation enzymes, and a moderate glycolytic

enzyme capacity.

Table 2-2. Bioenergetic enzyme activities in the costal diaphragm and two locomotor
muscles
Muscle CS HADH LDH Reference
Human COD 0.33 0.27 11.6 (163)
Rat COD (Rat) 0.46 0.23 4.8 (126)
PLA (Rat) 0.29 0.11 8.4 (126)
SOL (Rat) 0.33 0.22 2.5 (126)
COD, costal diaphragm; PL, plantaris; SOL, soleus; CS, citrate synthase; HADH, 3-
hydroxyacyl-CoA dehydrogenase; LDH, lactate dehydrogenase. Enzyme activities are
expressed as jiM/min/mg of protein.

Muscle Atrophy

The diaphragm, like all skeletal muscle, is highly plastic and therefore rapidly

adapts to the demands placed on it. Skeletal muscle quickly adapts to alterations in

proprioceptive activity, motor innervation, mechanical load, and joint mobility (10).

Skeletal muscle adapts to an increase in muscular activity by increasing contractile and

structural protein content (hypertrophy), whereas inactivity or disuse leads to a loss of

muscle mass (atrophy) (10). Hypertrophy (protein accumulation) and atrophy (net loss of

protein), therefore, are critically dependent on the relative rates of protein synthesis and

protein degradation (61). Atrophy results in a decrease in cross sectional area (CSA) and

this is functionally significant because muscle strength is directly related to CSA (26).

MV is a common method of reducing or removing diaphragmatic work. The

reduction in workload by MV leads to diaphragmatic disuse muscle atrophy and/or









weakness and is a major mechanism of weaning failure (167). The term disuse is relative

and can be defined as a reduced level of contractile activity (116). Two characteristic

components of reduced contractile activity include hypokinesia and hypodynamia (116).

Hypokinesia refers to a decreased level of contractile activity (i.e., reduced limb

movements) and hypodynamia is a decrease in mechanical loading (i.e., reduced weight-

bearing function) (116). MV and other models of skeletal muscle disuse atrophy are

reviewed below.

Models of Locomotor Muscle Atrophy

Several different experimental models and human clinical conditions result in

skeletal muscle atrophy. These models and conditions will be reviewed according to the

level of neuromuscular activation altered by electrical activation and weight-bearing

status.

Reduced Electrical Activation and Load Bearing

The electrical activation and load bearing status of skeletal muscle can be altered

by spinal cord injury, spinal cord transaction, and limb immobilization (with the muscle

of interest in the shortened position). In this section, spinal cord injury (SCI) and spinal

cord transaction (ST) will be considered together. Spinal cord transaction interrupts the

upper motor neuron pathway by transecting the spinal cord, often at the thoracic level. In

cat soleus, ST results in a 75% reduction in the daily-integrated electromyographic

(EMG) activity and a 66% reduction in the total duration of the EMG (1). After ST, the

ankle joint of the animal is held in an extended position, effectively unloading the soleus

(1). After 5 and 10 days of ST, the rat soleus significantly atrophies, -35% and 60%

reduction in the total CSA respectively (45). Further, ST alters myosin expression from

slow-to-fast MHC at both the mRNA (45) and protein level in the soleus (45, 138). Six to









8 months after ST biochemical measures such as citrate synthase and myosin ATPase

activities become characteristic of fast muscle (138). The functional outcome of ST is a

loss of absolute force generating ability by the affected muscless. The absolute tension

generation by the medial gastrocnemius and soleus after 6 to 8 months of ST is reduced

by 26% (137) and 50% respectively (138).

Human skeletal muscle also undergoes a slow-to-fast MHC shift After SCI. The

vastus lateralis from 15 patients was sampled 6, 11, and 24 weeks after SCI (33). At the 6

and 11 week time point there was no detectable MHC shift but after 24 weeks there was a

16% increase in type IIx MHC expression (33). Thus, human skeletal muscle adapts to

reduced electrical activation and muscle loading but at a slower rate than small animals

such as rat and cat. Overall, ST and SCI lead to alterations in fiber type composition and

impairs force generation.

Hindlimb immobilization (HI) is a clinically relevant model of reduced electrical

activity and loading. Hindlimb immobilization is achieved by fixing the joint(s) at a

specific angle by either pinning the joint(s) surgically with steel rods or by fixing the

limb with orthopedic plaster. The electrical activation of the rat soleus after HI in the

neutral position is -50% of control (52). Similarly, the electrical activity of the human

quadriceps is -60% less than the electrical activity observed in control quadriceps (77).

Atrophy induced by HI results in significant decrements in the CSA of both rat (9) and

human (77) skeletal muscle within 7 to 21 days, respectively. Accordingly, the decrease

in CSA results in an -50% loss of strength in rat soleus (177) and human quadriceps (77)

skeletal muscle. Metabolic markers such as citrate synthase and lactate dehydrogenase

indicate that HI shifts the normally slow oxidative soleus toward a fast oxidative









glycolytic muscle (51). Further, the slow-to-fast MHC shift occurs rapidly at the mRNA

level. After 1 week of HI soleus IIx and IIb mRNA transcript levels increased -24 and

2.6 fold compared to control (83). Fiber type conversion in human skeletal muscle After

HI also follows the slow-to-fast pattern. After 3 weeks of HI, type I mRNA transcripts

were -30% less than control and IIx transcripts were -300 times greater than control

(77). To date, there are no data on the levels of MHC protein isoforms after HI in either

the rat or humans.

Within hours of HI, protein synthesis and protein degradation are altered. Six hours

after HI the fractional rate of protein synthesis in the soleus is reduced 20-35% (28, 60).

For example, Goldspink (60) measured a 20% decrease in the synthetic rate with -2%

loss of muscle wet weight and a slight decrease in the rate of protein breakdown after 6

hours of HI. After 2 days of HI, soleus wet weight was reduced by 25%, the rate of

protein synthesis was 65% less, and the rate of protein degradation was 50% greater than

control (60). Booth (28) reported similar findings after 6 hours of bilateral HI, a -35%

decrement in the fractional rate of protein synthesis in the gastrocnemius.

Reduced Loading

Due to the limited nature of spaceflight, ground based models such as hindlimb

unloading (HU) of the rat, and human bed-rest models have been devised as models of

reduced loading.

Hindlimb unloading of the rat is typically achieved by placing a plaster cast at the

base of the tail to which a gimbal is attached. The animal is then raised such that the

forelimbs support the weight of the animal and its hindlimbs are not in contact with the

ground. To date, EMG recordings of rat hindlimb activity have not been made during

actual spaceflight. However, EMG recordings of rat soleus during parabolic flight, -25









sec of micro-gravity, is -10% of control values (95). This rapid decrease in EMG activity

has also been observed using HU. The EMG activity of the soleus is nearly abolished -3

sec after HU is imposed (5). Chronic soleus EMG activity either remains decreased (24)

or returns to normal (5) after 28 days of HU. While actual force measurements have not

been made during spaceflight or HU, it is generally accepted that force generation is

minimal (159). The contractions are spontaneous isotonic contractions and do not

generate much force because they are not weight bearing and are similar to the contractile

patterns that occur in the muscles of astronauts in space (27).

Spaceflight and HU rapidly affect muscle weight. After 5 to 7 days of spaceflight,

rat soleus mass is -18 to 30% less than control (75). Additionally, soleus muscle fiber

CSA is significantly reduced (-14%) and absolute tension is significantly impaired (-

28%) (75). Hindlimb unloading induces similar outcomes after 4 to 5 days, -15-30% loss

of soleus mass (101, 155). Seven days of HU significantly reduces type I fiber CSA, -

14%, and absolute tension, -30% (110). Lactate dehydrogenase activity is not altered by

spaceflight, but HU significantly decreases its activity, -27% (117). Conversely, citrate

synthase activity is not affected by either spaceflight or HU (117). While the unloading

effect significantly alters slow antigravity muscles such as the soleus, the morphometry

and function of the fast extensor digitorum longus is not impacted by 14 days of

spaceflight (154).

The change in MHC phenotype in the HU soleus is a very predictable slow-to-fast

shift. After 4 days the loss of type I MHC is small, but after 7 days the loss is significant

(155). The increase in type IIa MHC expression is significant after only 4 days (155).

After 7 days, type IId/x MHC is significantly increased and an increase in type IIb is









found after 14 days (155). The expression of each MHC mRNA transcript roughly

mirrors protein expression. A small change in type IP3 MHC mRNA (the mRNA

transcript for type I MHC) expression occurs after 4 to 7 days and a significant increase

in the expression of Icl mRNA (the transition mRNA transcript from I3 to IIa) occurs in

the same time period (155). Type IIa mRNA expression increases slightly and IId/x and

IIb expression significantly increases within 4 days (155). The decrease in type I and IIa

mRNA (>30%) and the increase in IId/x and IIb (>100%) mRNA becomes significant

after longer periods of time, 9-14 days in space (32, 67) and thus, the slow-to-fast MHC

shift rapidly occurs at both the protein and mRNA level.

A contributory factor leading to the loss of muscle mass under reduced loading

conditions is the decreased rate of protein synthesis. Total mixed protein synthesis and

myofibrillar protein synthesis measured during the first 5 hours of HU decreases 16% and

22% respectively in the rat soleus (162). In these experiments protein synthesis was

measured by constant infusion of radio-labeled leucine into the animal over the 5 hour

period. The measurement of protein synthesis during the first 5 hours of HU was

therefore an average of the initial response and may not reflect the true rate during the 5th

hour. However, after 24 hours of HU, total and myofibrillar protein synthesis in the

soleus is significantly diminished, -30 and -15% respectively (115). Thus, skeletal muscle

adapts rapidly to HU by decreasing protein synthesis and importantly, the rate of

myofibrillar protein synthesis.

Models of Inactivity

Inactivity includes spinal cord isolation and blockage of the motoneuron action

potential conduction by substances such as tetrodotoxin. Also,denervation is often









included as a model of inactivity. Denervation is achieved by severing the motoneuron-

muscle connection, but because it also interrupts neural input to tissues such as vascular

tissue, thus altering blood flow, its effect is often difficult to interpret (116) and will not

be considered here.

Spinal cord isolation (SI) has been used to study the effects of nueromuscular

activity on skeletal muscle. Spinal cord isolation involves complete spinal cord

transaction at the mid or low thoracic and lumbar-sacral levels, as well as complete

deafferentiation between the two points of transaction (159). This preparation maintains

the integrity of the neuromuscular unit and yet eliminates sensory input from the dorsal

roots as well as neural signals from either above or below the transaction (159).

The atrophic effect of SI is severe and rapid. The soleus loses 25% of its mass and

75% of its fiber CSA 4 days after SI (64). The slow-to-fast shift in MHC is relatively

slow, resulting in a 10% increase in IId/x expression after 15 days and a 40% loss of type

I MHC after 60 days (64). Spinal cord isolation causes skeletal muscle to become not

only smaller and faster, but weaker as well. Maximum force generation by the soleus 6

months after SI is decreased by 80% (136). The enzymatic profile of skeletal muscle

shifts from oxidative (-70% succinate dehydrogenase activity) toward glycolytic (+120%

glycerolphosphate dehydrogenase activity) after SI (84). To date, there are no published

reports of total and/or myofibrillar protein synthesis After SI, however, the rate and

severity of SI- induced atrophy suggests that both would be decreased.

Tetrodotoxin (TTX) is a potent Na+ channel blocker and if applied continually to

motor nerves it will inactivate skeletal muscle. Muscle paralysis after TTX treatment, as

determined by EMG activity during locomotion, is nearly complete in all treated animals









(152). This model of inactivity leads to significant atrophy, 46.5% decrease in muscle

mass and a 25% decrease in myofibrillar protein in 2 weeks (152). Concomitant with the

TTX-induced atrophy is a 38% loss of force generating ability (152). The expression of

MHC is altered by TTX treatment such that the expression of type I MHC increases, type

IIa decreases, type IId/x increases, and type IIb does not change (112). It is currently

unknown why type I MHC expression increases in response to TTX treatment but it may

be related to the disruption in the delivery of a neurotrophic factor to the motor end-plate

(159). Tetrodotoxin treatment severely diminishes the metabolic capacity of both

oxidative (citrate synthase -33%) and glycolytic (phosphfructokinase -70% and ca-

glycerolphosphate dehydrogenase -58%) enzymes (153). The effect of TTX treatment on

protein synthesis is currently unknown but it likely plays a role in the observed atrophy

response.

Models of Atrophy in the Diaphragm

The primary muscle of inspiration is the diaphragm and it is chronically active

throughout life. Due to its chronic activity the activation pattern of the diaphragm differs

from locomotor muscles such as the extensor digitorum longus (EDL) and the soleus. The

rat diaphragm has a duty cycle (duration of inspiratory time/total respiratory cycle

duration) of -40% (141) while the EDL and soleus have duty cycles of 2% and 14%

respectively (71). Thus, the contractile history of the diaphragm differs greatly from

locomotor muscles and it may be more susceptible to alterations caused by inactivity

(113). Models of diaphragmatic inactivity include denervation, blockade of nerve

impulses by TTX, spinal cord transaction (ST), and MV. The effects of MV will be

considered separately from the other models.









Procedure for Investigating Diaphragmatic Atrophy

Diaphragmatic denervation (DNV) is accomplished by dissecting the phrenic

nerve, unilaterally or bilaterally. Once dissected, the phrenic nerve is transected

(phrenicectomy) and a significant portion is removed (e.g. -10-20 mm in the rat) to

prevent possible reinnervation of the diaphragm (98).

Again, TTX is a Na+ channel blocker that prevents action potential propagation.

The phrenic nerve is typically dissected at the lower neck and a Silastic cuff is placed

around the nerve and connected to a miniosmotic pump that continuously perfuses the

nerve with TTX.

Spinal cord transaction (ST) is achieved by performing a dorsal laminectomy after

which one-half (e.g. right side) of the cervical spinal cord (e.g. at C2) is sectioned from

the dorsal root to the ventral root. Correctly done, only the ventral and lateral funiculi are

cut and the lateral funiculus is preserved in order to minimize motor deficits in the

ipsilateral side (113).

Electromyographic Activity

Paralysis of the diaphragm is verified by the EMG activity of the left and right

sides (hemidiaphragm) of the diaphragm. The EMG signal is obtained by implanting

small diameter wire electrodes into the diaphragm. Each of the above-mentioned surgical

preparations induces diaphragmatic paralysis (i.e., no EMG activity) of the intended

hemidiaphragm, and a 50% increase in the activity of the intact hemidiaphragm due to a

compensatory increase in muscle activation (183). However, phrenic nerve activity after

unilateral DNV and unilateral TTX significantly increases -40-50% on both sides (113).

The increase in activity indicates that there is a compensatory increase in central drive to

motor neurons on both sides of the spinal cord (113). The ST model, however, leads to









inactivity of the phrenic motoneurons (113). Diaphragm paralysis and motoneuron

activity are matched in the ST model, whereas motoneuron activity and paralysis are not

matched in the TTX model, and the connection between the nerve and muscle is

obviously severed in the DNV model (113). The morphological, biochemical, and

mechanical alterations that result from each of the above models is attenuated when the

activity of the diaphragm muscle fibers is matched by the phrenic motoneuron.

Diaphragmatic Mass

The initial response to diaphragmatic paralysis via DNV is hypertrophic after

which the response becomes atrophic. After 8 days of unilateral and bilateral DNV, the

diaphragm hypertrophies 20% (paralyzed hemidiaphragm) and 13% (each

hemidiaphragm hypertrophied) respectively (179). The hypertrophic response is

diminished by the second week and diaphragmatic mass returns normal (185). The

chronic response (i.e., 6 weeks) to diaphragmatic paralysis is a significant loss of

diaphragmatic mass (-37%) (98). To date, the effect of ST and TTX on diaphragmatic

mass has not been determined.

Differential Fiber Type Response to Diaphragmatic Inactivity

The change in cross-sectional area (CSA) after unilateral hemidiaphragm paralysis

induced by ST, TTX, and DNV is variable; in each case the alterations brought about by

ST are not as dramatic as those of TTX and DNV after 2 weeks of treatment. Type I fiber

area hypertorphies 33, 70 and 80% according to treatment, ST, TTX, and DNV,

respectively (184). Type IIa fibers also hypertrophy 13, 99, and 81% after ST, TTX, and

DNV respectively, (184). Paralysis induced by TTX and DNV leads to the appearance of

hybrid type I/IIa fibers (184). Type IId/x fiber CSA is reduced 5, 34, and 39% and type

IIb fibers also atrophy 15, 53, and 57% after 2 weeks of ST, TTX, and DNV respectively









(184). The variable response to the treatments may be the result of better matching

between muscle fiber and motoneuron activity in the ST model. After 6 weeks of

unilateral DNV, the CSA of the type I and IIa fibers returns to normal, whereas the IIb/x

fibers lose -57% of their CSA (98). The hybrid I/IIa fibers that appear after 2 weeks of

DNV (184) occupy -50% of the total CSA after 6 weeks of DNV (98), indicating a slow-

to-fast shift in MHC.

Myosin Heavy Chain Content

Diaphragmatic MHC content has been followed over a 3-week period of DNV

(147). Type I MHC increases after 1 week (+30%) and begins to decrease during the 2nd

(+25%) and 3rd week (-6%) (147). The type IIa MHC expression is elevated during the

first 2 weeks, +23% week one and +29% week two, and begins to return to normal by the

3rd week, +11% (147). The expression of type IId/x MHC falls off rapidly during the

first 2 weeks, -27% and -24%, and then begins to return to normal by week three, -6%

(147). Type IIb MHC expression declines rapidly and remains depressed over the entire

3-week period, -50% to -66% (147).

Myosin Heavy Chain mRNA

Northern analysis of MHC mRNA reveals a down regulation of all 4 MHC

transcripts after 8 days of DNV (179). Type I, IIa, IId/x, and IIb mRNA levels

significantly decrease 50, 70, 60, and 35% respectively after 8 days of reduced activity

(179). This is an interesting observation when one considers the finding that type I and

IIa MHC content increases and IId/x and IIb expression decreases at the protein level

(147). These observations suggest differential posttranscriptional regulation of the four

MHCs.









Contractile Properties

After 2 weeks of unilateral hemidiaphragm paralysis induced by ST, TTX and

DNV, specific tension declines 23, 49, and 51% respectively, (113). In accordance with

the observed, type I and IIa hypertrophy (184) and the increase in type I and IIa MHC

expression after diaphragmatic paralysis, diaphragmatic contraction and relaxation times

dramatically increase (113). Additionally, actomyosin ATPase activity is reduced by all

three treatments (184). Again, the effects of ST were not as pronounced as those induced

by TTX or DNV (113, 184).

Oxidative Capacity

Succinate dehydrogenase (SDH), a marker of oxidative capacity, is significantly

reduced after 2 weeks of ST, TTX and DNV (184). The reduction in SDH activity did not

occur in all fiber types however. The oxidative capacity was not altered in the type I or

IIa fibers. Paralysis induced by TTX and DNV reduced SDH activity in type IId/x and IIb

fibers whereas ST only reduced SDH activity in the IId/x fibers (184).

Protein Synthesis

To date, measures of protein synthesis have only been performed using the DNV

model of disuse. As might be expected, in vivo protein synthesis is significantly increased

(-50%) during the initial hypertrophic phase, days 1 through 10 (169). The significant

increase in protein synthesis after acute DNV has also been observed under in vitro

conditions (170). However, after approximately 3 weeks of DNV, the diaphragm

atrophies significantly (29). While the rate of protein synthesis has not been measured

after chronic diaphragm DNV, measures of diaphragmatic total RNA content have been

made. During the hypertrophic period as well as the atrophic period, followed out for 51

days, total RNA content in the diaphragm parallels diaphragmatic mass (29). The









decrease in total RNA content and the loss of diaphragmatic mass suggests that protein

synthesis would be suppressed.

Diaphragm Length Changes

It has been hypothesized that passive stretch is an underlying mechanism

responsible for the morphological adaptations incurred by the paralyzed side of the

diaphragm (183). The paralyzed hemidiaphragm is "pulled" along by the intact

hemidiaphragm as it contracts. Due to differences in muscle fiber orientation, the length

changes that occur after unilateral paralysis varies between regions of the diaphragm. The

muscle fibers in the paralyzed midcostal region of the diaphragm are stretched -3-5%

beyond resting length (diaphragm muscle fiber length at end expiration) because they are

in series with the muscle fibers in the intact hemidiaphragm. The sternal region is

passively shortened 4-5% of resting length because the muscle fibers are in parallel with

the fibers of the opposite side (183). However, the degree of adaptation, as measured by

in vitro contractile properties and fiber typing, was similar between regions, costal and

sternal. If alteration of muscle fiber length, stretching or shortening, was the underlying

cause for the observed changes in in vitro contractile properties after DNV, a differential

response (e.g., passive shortening would induce greater contractile dysfunction) would be

predicted. This was not the case and the authors suggest that removal of innervation itself

is the underlying mechanism leading to the morphological adaptations (183). This

observation is supported by earlier findings that reported less pronounced morphological

and contractile adaptations after ST as compared to TTX or DNV (184). This suggests

that interactions between the motoneuron and muscle fibers may play an integral role in

muscle adaptation (183).









Mechanical Ventilation and Diaphragmatic Atrophy

MV is used to either fully support or augment alveolar ventilation. Further, MV

"rests" the diaphragm and in doing so the phasic activity of the diaphragm is decreased

by varying degrees, depending on the level of mechanical support.

Electrical Activity

Inhibition of diaphragmatic EMG activity during controlled MV (CMV) has been

demonstrated in healthy subjects (91, 107, 148, 149) and in chronic obstructive

pulmonary disease (COPD) patients (30). Likewise, MV reduces neuronal activity in

regions of the brain that are known to be involved in the control of breathing (48).

Importantly, the loss or reduction of EMG activity of a muscle is a primary factor in the

etiology of disuse atrophy (e.g., 45, 52).

Diaphragmatic Disuse Atrophy

Mechanical ventilation is frequently used in caring for neonates and infants. The

CSA of muscle fibers from the diaphragms of neonates and infants ventilated for more

than 12 days before death is markedly smaller, -70% in one case, and consistent with

disuse atrophy (86). Diaphragmatic atrophy may also be observed after cervical fracture.

Patients with lesions above the origins of the phrenic nerve roots require ventilatory

assistance (e.g., MV or phrenic nerve pacing). Measures of diaphragm thickness have

been performed on patients treated with phrenic nerve pacing. The normal adult

diaphragm is -0.3 cm thick (109) and after 8 months of MV the thickness can decrease to

-0.18 cm (14). After 6 weeks of phrenic nerve pacing, diaphragmatic thickness can

increase nearly two-fold with a three-fold improvement in maximal tidal volume (14).

While this is not conclusive evidence of diaphragmatic atrophy induced by prolonged

MV, it is highly suggestive of diaphragmatic atrophy.









Animal Models and Mechanical Ventilation

Due to the invasiveness of intubation and diaphragmatic biopsies, animal models

have been devised to study the mechanical and biochemical effects of MV on the

diaphragm. Anzueto et al. (8) tested the hypothesis that prolonged MV would impair

diaphragmatic contractile properties. The investigators ventilated healthy adult baboons

for 11 days and measured maximal transdiaphragmatic pressure (Pdimax), the force

frequency response, and endurance time pre- and post-MV (8). The ability of the

diaphragm to generate force in vivo can be determined by measuring Pdimax. This

measurement of diaphragmatic strength was impaired by 11 days of MV, -32% to -48%.

The diaphragmatic response to phrenic nerve stimulation over a wide range of

frequencies (i.e., the force frequency response) was dramatically shifted downward after

11 days of MV (8), indicating a loss of force generating ability across a spectrum of

stimulation frequencies. Fatigue resistance was assessed by requiring the animals to

breath through a resistor at 60-70% of Pdimax until the target pressure could no longer be

met. Diaphragmatic endurance time was reduced by -36% after 11 days of MV (8).

It is possible that the use of a neuromuscular blocking agent during the MV period

contributed to the deleterious effects of MV. The investigators did however control for

this by withholding the neuromuscular blocking agent for 8 hours before contractile

measurements were made. Contractile measurements were conducted only after

spontaneous breathing resumed and the diaphragm responded to phrenic nerve

stimulation (8). Typically, muscle weakness occurs when neuromuscular blocking agents

are used in conjunction with corticosteroids (36, 93) and only rarely occurs in patients not

treated concurrently with corticosteroids (59). Nonetheless, the decrease in diaphragmatic









force generation is similar to the results of Le Bourdelles et al. (92) and Powers et al.

(125) who did not use neuromuscular blocking agents to study the effects of MV.

The rat has also been used to investigate the impact of MV on the diaphragm. After

48 hours of MV, the mechanical properties of the diaphragm, soleus, and EDL as well as

their biochemical properties have been documented (92). MV did not significantly alter

body mass but the mass of the diaphragm, soleus, and EDL were significantly decreased

after 48 hours of MV (92). MV did not affect the contractile properties of the soleus or

EDL (92). However, diaphragmatic contractility was significantly impaired by 48 hours

of MV. MV resulted in a downward shift in the force frequency response and reduced

maximal tetanic tension -60% (92). Further, MV negatively impacts indicators of

calcium handling within the muscle such as the rate of force development (dP/dt) and the

rate of relaxation (92). The slowing of these indicators suggests a decreased rate of

calcium release and sequestration. MV did not affect the bioenergetic enzymes, citrate

synthase and lactate dehydrogenase in the diaphragm (92). This indicates that the

observed diaphragmatic dysfunction is not necessarily due to a derangement of

metabolism but rather, an alteration in one or more steps of excitation-contraction

coupling.

Effects of Mechanical Ventilation on Diaphragmatic Contractile Properties

Recently, our laboratory characterized the time course of MV induced

diaphragmatic dysfunction (125). Animals were mechanically ventilated in the control

mode for 12 to 24 hours. At the specified time point, a segment of the costal portion of

the diaphragm was carefully dissected and used for in vitro contractile measurements.

The body mass of each animal was measured pre- and post-MV and there was no change

(p > 0.05). Additionally, the mass of the soleus did not differ between control and MV









animals (p > 0.05). Arterial blood pressure, blood gases, and pH status were monitored

throughout the MV period and were found to fluctuate, but remained within a narrow

physiological range. Maintenance of blood gas and pH homeostasis is critical because

hypoxia, hypercapnia, and respiratory acidosis are known to impair diaphragmatic

function.

Our findings reveal that contractile dysfunction occurs in as few 12 hours. Twitch

tension was 35% less than control after 12 hours of MV. Twitch tension continued to fall

over the 24-hour period; at which point, the twitch tension of the mechanically ventilated

diaphragms was 42% less than control. The force frequency response of the mechanically

ventilated diaphragms was impaired, shifted to the right, at all stimulation frequencies.

The magnitude of the right-shift was exacerbated with time on the ventilator. MV

significantly reduced (p < 0.05) maximal specific tension and the loss of force generating

ability fell over time (i.e., -18% after 12 hours and -46% after 24 hours of MV).

Our results of a 46% loss of specific tension are comparable to those of Le

Bourdelles et al. (92) who reported a 60% loss of specific tension after 48 hours of MV.

Further, our results indicate that the effect of MV is confined to the diaphragm as there

was no loss of soleus muscle mass. This observation is similar to the finding of Le

Bourdelles et al. (92) who reported no change in soleus or EDL maximal specific tension

after 48 hours of MV.

Mechanical Ventilation and Diaphragmatic Atrophy

After the time course experiments, we tested the hypotheses that short-term MV

(18 hours) would induce atrophy in all four fiber types in the diaphragm, increase the rate

of diaphragmatic muscle protein degradation, and increase oxidative stress in the

diaphragm (145).









After 18 hours of MV there was no loss of body or soleus mass. However, total

diaphragmatic mass was significantly reduced (-6.9%) and this was primarily due to the

significant loss of costal diaphragmatic mass (-7.3%) (145). The atrophic effect of 18

hours of MV was confirmed using immunohistological techniques: the CSA of the type I

fibers was reduced -15%, IIa -27%, IId/x -30%, and IIb -24% (145). Again, these

findings support the postulate that MV results in diaphragmatic atrophy.

Measures of total and myofibrillar protein content were made to determine if the

observed contractile dysfunction and decreased diaphragmatic mass could be explained

by alterations in the protein composition of the diaphragm after MV. Eighteen hours of

MV resulted in significant reductions in diaphragmatic protein. Specifically, the

concentration of both myofibrillar protein and soluble protein significantly decreased by

-10%, resulting in a significant decrease in the total protein concentration (145).

Consistent with the loss of diaphragmatic mass, was the reduction in total (-16%) and

myofibrillar protein content (-16%), reflecting an absolute loss of protein from the

diaphragm (145). Additionally, MV resulted in a mean increase (-4%) in muscle water

content (145).

The rate of protein degradation was also measured in vitro (145). After 18 hours of

MV two strips from the costal diaphragm were removed and suspended in separate in

vitro tissue chambers filled with a modified Krebs solution and aerated with 95% 02/ 5%

CO2 for 2 hours. The tyrosine concentration in the bathing medium was subsequently

analyzed fluorometrically (172). The rate of tyrosine release was used to determine the

rate of total protein catabolism because this amino acid is neither synthesized nor

degraded by skeletal muscle (164). The significant loss of protein, was due in part to the









significant increase in protein degradation, as indicated by a 28% increase in tyrosine

release (145).

Our results also revealed that MV results in an increase in diaphragmatic oxidative

stress (145). The diaphragmatic content of both total 8-isoprostane and protein carbonyls

increased 30% and 35% respectively (145). Tissue levels of total 8-isoprostane and

protein carbonyls were measured as markers of lipid peroxidation and protein oxidation,

respectively. In the context of MV-induced diaphragmatic atrophy, an increase in protein

oxidation could be important because moderately oxidized proteins are more sensitive to

proteolytic attack by proteases (37, 38, 40, 96, 97, 118). Therefore, oxidative

modification of proteins could contribute to the elevated protein degradation measured

after MV. Further, the ubiquitin-proteosome pathway is the proteolytic pathway

implicated in the degradation of actin and myosin in muscle (55, 158) and this pathway is

up-regulated during periods of oxidative stress (142, 146, 161). An oxidative stress-

mediated up-regulation of the ubiquitin-proteosome pathway would lead to an increase in

protein degradation and thus atrophy.

Summary of Literature Review

MV is used to sustain patients with a life threatening illnesses, provide ventilation

during respiratory muscle failure ("respiratory muscle rest") or to compensate for a

damaged upper airway or to aid during recovery or rehabilitation from an illness.

Excessive inspiratory muscle workload due to obesity, asthma, or pulmonary resection

can lead to respiratory muscle failure and require MV to maintain the life of the patient.

There are many modes of MV but controlled MV (CMV) results in complete

diaphragmatic inactivity and may be an ideal model to study the effects of MV. While

CMV provides the maximum degree of respiratory muscle rest it is not without









consequence to the very muscle it is intended to aid, the diaphragm. The reduced use of

the diaphragm while receiving MV may lead to atrophy and increase the likelihood of

diaphragmatic weakness. If the weakness is substantial it may be difficult to wean the

patient. The process of weaning is challenging and contributes to a large portion of the

ICU workload.

The diaphragm is the primary muscle of inspiration and is chronically active

throughout life. The fiber type composition and the metabolic properties of the

diaphragm reflect the demand of this chronically active tissue. Studying the diaphragm is

often difficult to do using humans but the rat diaphragm has characteristics similar to the

human diaphragm, thus enabling difficult research questions to be addressed using this

model.

The diaphragm, like all skeletal muscle, is highly plastic and therefore rapidly

adapts to its demand. Skeletal muscle hypertrophies when muscular activity increases,

whereas inactivity or disuse leads atrophy. Hypertrophy and atrophy, are therefore,

critically dependent on the relative rates of protein synthesis and protein degradation.

Atrophy results in a decrease in CSA and is functionally significant because muscle

strength is directly related to CSA.

A loss or decrease in neural stimulation of a muscle results in atrophy. There are

various models used in the study of hindlimb muscle atrophy (e.g., HI, spaceflight and

HU, and ST). Postural locomotor muscles rapidly adapt to the reduced load. Markers of

this adaptation include a loss of mass and CSA, slow-to-fast shift in MHC, and a

metabolic shift toward a more glycolytic fiber. The functional significance of these

adaptations is a loss of strength and endurance.









Manipulation of the phrenic nerve via denervation (phrenicectomy), tetrodotoxin,

or spinal cord isolation initially results in diaphragmatic hypertrophy followed by

atrophy. Paralyzing the diaphragm results in a significant decrease in specific tension

during all stages of its adaptation to paralysis. Attenuation of the contractile and

biochemical changes when the inactivity of the muscle fibers is matched to the inactivity

of the phrenic motoneurons, spinal cord isolation model, is an intriguing finding.

MV, in particular controlled MV, eliminates diaphragmatic EMG activity. The loss

of diaphragmatic electrical activity leads to a decrease in force generation and impairs

endurance. Specifically, MV leads to a loss of both in vivo and in vitro force generating

ability. The impairment in force generation is exacerbated by time spent on the ventilator.

The significant loss of diaphragmatic mass (i.e., atrophy) occurs in as few as 18 hours.

The loss of diaphragmatic mass includes the loss of both total and myofibrillar protein

and this is due in part to an increase in proteolysis.














CHAPTER 3
METHODS

The methods section is organized according to the objectives of each specific aim,

animals and experimental design, and the methods used.

Experimental Design-Specific Aim #1

The effect of MV on protein synthesis was investigated by testing the hypothesis

that MV-induced diaphragmatic atrophy is due, at least in part, to a decreased rate of total

(mixed muscle protein (MMP)) and myosin heavy chain (MHC) protein synthesis.

Preliminary experiments indicated that prolonged MV results in significant

diaphragmatic atrophy. Both MMP and MHC protein synthesis rates (in vivo) in the

diaphragms of control, spontaneously breathing, and MV animals during 0 to 6, 6 to 12,

and 12 to 18 hours of MV were measured to determine the time course of changes in

protein synthesis.

Animals and Experimental Design

To address this specific aim, healthy young adult (4-month-old) female specific-

pathogen-free (SPF) Sprague-Dawley rats were individually housed and fed rat chow and

water ad libitum while being maintained on a 12 hour light/dark cycle for 3 weeks before

initiation of these experiments. Animals were randomly assigned to either the control or

MV experimental group. The control group was subdivided into four groups, control, or 6

to 18 hours of spontaneously breathing (SB 6, SB 12, SB 18). The control animals (n =

10) were not mechanically ventilated nor did they receive infusion of [1-13C]leucine. This

group was used to determine the natural abundance of [1-13C]leucine in the Sprague-









Dawley rat diaphragm. The SB 6 group (n = 10) was not mechanically ventilated but they

were infused with [1-13C]leucine while spontaneously breathing and anesthetized for 6

hours. Animals assigned to the SB 12 group (n = 10) were anesthetized for a total of 12

hours and [1-13C]leucine was infused during hours 6 tol2. Similarly, the SB 18 group (n

= 10) was anesthetized for a total of 18 hours and [1-13C]leucine was infused during

hours 12 to 18. The SB groups served as time matched controls for the MV groups.

The MV group was subdivided into three groups, MV 6, MV 12, and MV 18. The

MV groups were mechanically ventilated and the rate of protein synthesis was

determined after 6 (n = 10), 12 (n = 10), and 18 (n = 10) hours ofMV. [1-13C]leucine was

infused during the last 6 hours of the experimental period (i.e., 0 to 6, 6 to 12, and 12 to

18 hours).


Design

Control (n=10) SB 6 (n=10) SB 12 (n=10) SB 18 (n=10) MV6(n=10) MV 12 (n=10) MV 18 (n=10)
no MV (acute) no MV no MV no MV
no cl3Leu cl3Leu infusion cl3 Leu infusion cl3 Leu infusion cl3Leu infusion cl3Leu infusion cl3Leu infusion
during hrs 0-6 during hrs 6-12 during hrs 12-18 during hrs 0-6 during hrs 6-12 during hrs 12-18




Measure
[l-13C]leucine
incorporation


Figure 3-1. Experimental design for Specific Aim #1.

Mechanical ventilation protocol

Thirty minutes before anesthesia, animals received glycopryyloate (0.04 mg/kg IM)

in order to reduce airway secretions. Glycopryyolate was then administered every 2 hours

(0.04 mg/kg IM) for the remainder of the experiment. The animals were anesthetized









with an intraperitoneal (IP) injection of sodium pentobarbital (50 mg/kg of body weight).

Sodium pentobarbital was used as the general anesthetic because Le Bourdelles et al. (92)

have shown that the level of barbiturate required to maintain general anesthesia in rats

does not alter locomotor muscle contractile or biochemical properties. Additionally,

prolonged use of sodium pentobarbital (up to 18 hours) in rats does not induce atrophy in

locomotor muscles (i.e., soleus) (145). All surgical procedures were performed under

aseptic conditions. Once anesthetized, MV animals were tracheostomized and

mechanically ventilated with a volume-cycled ventilator (Inspira, Harvard Apparatus,

Cambridge, MA). A major advantage of volume-cycled ventilators is that tidal volume

remains relatively constant despite possible pathophysiological changes (e.g., airway

obstruction due to mucus secretion). Further, volume-cycled ventilators are capable of

maintaining a constant inspired percentage of oxygen (FIO2) which is important in

maintaining blood gas homeostasis during MV (20, 72, 165).

Heart rate and electrical activity of the heart was monitored via a lead II ECG using

needle electrodes placed subcutaneously. An arterial catheter was placed in the carotid

artery for constant measurement of blood pressure and blood samples (1 mL) were drawn

for analysis of [1-13C]leucine. Finally, a venous catheter was placed in the jugular vein to

add fluids and sodium pentobarbital.

Anesthesia was maintained during MV by intravenous infusion of sodium

pentobarbital (10 mg/kg body weight). Body (rectal) temperature was maintained at 370C

+10C by use of a computer controlled re-circulating heating blanket. Throughout MV

body fluid homeostasis was maintained via the administration of an intravenous

electrolyte solution, 2 mL/kg/hour. Continuing care during MV included expressing the









bladder, removal of airway mucus, lubricating the eyes, rotating the animal, and passive

movement of the limbs. Animals were continuously monitored during MV and while

spontaneously breathing, see below.

Postmortem examination

A board-certified pathologist (Dr. Sunjoo Kim, M.D.) performed all postmortem

procedures. Dr. Kim has extensive necropsy experience and is experienced in the clinical

microbiology techniques used to assess pneumonia and septicemia.

Necropsy examination. Necropsy examination included a detailed visual inspection

of the respiratory tract, the lungs, and the peritoneal cavity. Visualization of an abscess or

pus was considered a marker of infection, however no animals were infected.

Blood culture. Blood culture procedures were preformed according to the methods

recommended by the American Society for Microbiology (21) and Bailey and Scott's

Diagnostic Microbiology (82). A blood sample (0.5 mL) was drawn from each animal at

the conclusion of the experimental period via the jugular vein. Each sample was

immediately inoculated directly into the MacConkey agar plate (82). The blood cultures

were incubated at 370C for 5 days and inspected daily (82). Observation of bacterial

growth on the culture media would have been considered evidence of sepsis.

Control animals (nonmechanically ventilated) protocol

The animals in the control groups were randomly placed into one of four groups:

Control (acute anesthesia, no MV, no [1-13C]leucine infusion) or SB 6, SB 12, or SB 18

(anesthetized, spontaneously breathing, [1-13C]leucine infusion). Control animals (acute

anesthesia) were free of intervention during the hours before removal of the diaphragm

for measurement of biochemical properties. That is, these animals were not mechanically

ventilated before study. Control animals received an IP injection of sodium pentobarbital









(50 mg/kg body weight); after a surgical plane of anesthesia was reached their

diaphragms were removed for subsequent measurements of biochemical properties. SB 6,

SB 12, and SB 18 animals received the same surgical intervention and [1-13C]leucine

infusion paradigm as the MV animals except these animals were not mechanically

ventilated (i.e., they were breathing on their own during the entire experimental period).

The MV and SB animals received the same continuing care during the experimental

period and post mortem examination after the experimental period.

Methods Used: Biochemical Assays

Tissue removal and storage

At the appropriate times (as specified in the experimental design) biochemical

studies were conducted on muscle samples taken from the costal portion of the

diaphragm. Costal diaphragm segments obtained for biochemical analysis were rapidly

frozen in liquid nitrogen and stored at -800C until assay.

Rates of in vivo diaphragmatic protein synthesis

Infusion. The fractional rate of diaphragm muscle protein synthesis was measured

using intravenous infusion of [1-13C]leucine (Cambridge Isotopes Laboratory, Andover,

MA) and quantifying the in vivo rate of incorporation of [1-13C]leucine into both total and

contractile proteins in the diaphragm by gas chromatography-combustion-isotope ratio

mass spectrometry (GC-C-IRMS). The stable isotope [1-13C]leucine was chosen for

several reasons:

* Less isotope effect (i.e., less tissue injury).
* Small tissue sample is required for analysis.
* Safety.
* Validity and reliability of this label for measurement of the rate of in vivo muscle
protein synthesis is well established (121).









Animals were anesthetized with sodium pentobarbital and a tygon catheter was

placed in the jugular vein for infusion of [1-13C]leucine. A polyethylene catheter was also

placed in the carotid artery for blood sampling. Note, the jugular and carotid catheters

were the same as described above for adding electrolyte solution and sodium

pentobarbital and for monitoring arterial blood pressure. The jugular vein and carotid

artery were catheterized in the SB and MV groups immediately after anesthetization. The

catheter was kept patent by periodically flushing with heparinized saline (20 U/mL). At

the beginning of the infusion, animals were primed with 1.6 mg [1-13C]leucine/100 grams

of body weight, followed by a constant infusion rate of 0.20 mg[l-13C]leucine/100 grams

of body weight/ hour for 6 hours (KD Scientific Model 100 syringe pump, Boston, MA).

This infusion rate was chosen on the basis of previous experiments indicating that this

rate results in optimal muscle [1-13C]leucine enrichment for the determination of protein

synthesis in rat skeletal muscle (181). The infusion of the labeled amino acid is delivered

such that -5-10% of the plasma pool of free amino acids is labeled (130). In this sense

the labeled amino acid acts as a tracer with little or no effect on the overall metabolism of

tissue being investigated (130). The duration of the infusion period need only be long

enough for enrichment of the target protein but not so long as to allow recycling of the

tracer (130). Skeletal muscle protein turns over slowly and thus tracer recycling does not

present a problem (130).

Immediately before infusion, and at the end of the 5th and 6th hours of infusion,

blood samples (1 mL) were drawn for the measurement of plasma a-ketoisocaproate (a-

KIC) enrichment. At the completion of the experimental period, the costal diaphragm was

rapidly removed, frozen in liquid nitrogen, and stored at -800C until assay. Note that the









infusion pump continued to infuse at the time of tissue removal. A tissue sample only

needs to be taken at the end of the infusion period if no other labeled amino acid has been

administered previously. The baseline sample labeling can be assumed to be close to

previously measured tissue from the same tissue population (57) or from mixed blood

protein (73).

Sample analysis and calculations. Plasma a-KIC was isolated, prepared as the

trimethylsilyl quinoxalinol derivative, and analyzed for [1-13C]leucine abundance by use

of gas chromatography-electron impact quadrupole-mass spectrometry (GC-MS) (HP

5890 Series II and GC/HP 5970 Series MS, Hewlett-Packard, Avondale, PA). Tissue

fluid-free amino acids were extracted by homogenization in 10% TCA. The N-

heptafluorobutyryl propyl esters were formed, and the [1-13C]leucine abundance was

determined using electron capture-negative chemical ionization GC/MS (HP 5988A

Series II and GC/HP 5970 Series MS, Hewlett-Packard). The plasma a-KIC enrichment

and the tissue fluid [1-13C]leucine enrichment [in mole percent in excess (MPE)] were

used to represent the precursor pool (leucyl tRNA) enrichment for the calculation of

protein synthesis rate (Ks; the percentage of the protein mass synthesized per hour

(%/hour)). The following equation was used to calculate Ks

[1 -13 C] leucine MPE enrichment in protein x 100
K s =---------------
precursor pool enrichemnt x (t, t )

where (t2-tl) is the infusion time (hours).

The rate of protein synthesis can be determined if the site of the incorporation of

the amino acid into protein (i.e., in the appropriate aminoacyl-tRNA pool, is known)

(130). However since identification of the correct pool presents a problem (130), a

surrogate index of the labeling of the aminoacyl-tRNA pool has been adopted. The









branched chain amino acid leucine is the preferred tracer and the labeling of its

transamination product, a-ketoisocaproate (c-KIC), is measured. The use of a-KIC as

the surrogate of the true precursor labeling is possible because the formation of its

transamination product, a-KIC, occurs intracellularly and thus a-KIC reflects the extent

of labeling in the true precursor pools for protein synthesis (130). Indeed, the labeling of

a-KIC after infusion of labeled leucine is within 10% of the labeling of tRNA in human

skeletal muscle (174).

Analysis of mixed muscle protein synthesis rates. To determine the [1-13C]leucine

abundance in mixed muscle protein (MMP) 50-60 mg muscle samples were homogenized

in 1 mL of 10% TCA and hydrolyzed in 6 N HC1 at 1100C for 24 hours. The n-acetyl n-

propyl (NAP) esters of the component amino acids were formed, and the [1-13C]leucine

abundance in the hydrolyzed MMP were determined using GC-C-IRMS according

Yarasheski et al. (181).

Isolation of MHC for analysis of synthesis rates. All procedures for the MHC

extractions were performed on ice or at 40C. Frozen muscle samples (60-80 mg) were

homogenized in 1 mL of a 250 mM sucrose buffer (in mM: 250 sucrose, 100 KC1, 5

EDTA, and 20 imidazole, pH 6.8). The homogenate was centrifuged at 1,200 x g for 10

minutes, and the supernatant was discarded. The pellet was suspended in 1 mL of a 0.5%

Triton X-100 solution (175 mM KC1, 0.5% Triton X-100, pH 6.8), a modification of

Solaro et al. (150). The suspension was homogenized and centrifuged as before. This step

removes many of the soluble matrix proteins. The resultant pellet was rinsed (i.e.,

homogenized and centrifuged) with 1 mL wash buffer (150 mM KC1 and 20 mM Tris, pH

7.0) to remove excess Triton X-100 solution. The pellet was then frozen at -800C. The









pellet was resuspended in SDS buffer (62.5 mM Tris, 2% SDS, 10% glycerol, 0.001%

bromophenol blue, and 5% P-mercaptoethanol) and boiled for 5 minutes.

These extracts of myofibrillar protein (containing ~1 mg) were separated by SDS-

PAGE. All electrophoresis chemicals were purchased from Bio-Rad (Hecules, CA). Each

extract was separated on an individual gel with a single wide lane, utilizing a 7% T-2.5%

C polyacrylamide slab gel with a 4% T-2.5% C stacking gel. The proteins were separated

(150 volts, -5 hours) using a discontinuous buffer system that useed a Tris-Tricine buffer

(1.6 mM Tris, 16 mM Tricine, 0.01% SDS, pH 6.4) and a Tris buffer (2.5 mM Tris,

0.01% SDS, pH 6.4) as the cathode and anode buffers, respectively.

The separated protein was visualized by Coomassie staining (0.1% Coomassie

brilliant blue R-250, 45% methanol, and 10% glacial acetic acid) for 10 to 15 minutes.

after overnight destaining (30% methanol and 10% glacial acetic acid), the band

corresponding to the molecular mass of MHC was identified. The protein band was

carefully cut out within the distinctly stained boundary, minced, and put into a tube. The

samples were hydrolyzed in 3 to 4 mL of concentrated HC1 (110C for 48 hours), and the

NAP esters of the amino acids were prepared for analysis of [1-13C]leucine abundance

using GC-C-IRMS.

Measuring mixed muscle (MMP) and MHC protein synthesis is very involved but

the method is well established and in everyday use in the Yarasheski laboratory. The

method of accurate identification of the MHC band, the quantity and quality of the band,

and the reliability of the isotopic enrichments are well documented (70).









Statistical analysis

Planned comparisons were made between relevant groups with a Bonferroni

correction for the number of comparisons conducted. Significance was established at

p < 0.05.

Experimental Design-Specific Aim #2

The effect of MV on MHC mRNA content was determined by testing the

hypothesis that MV alters pretranslational events in the diaphragm. Preliminary

experiments indicated that prolonged MV results in significant diaphragmatic atrophy

and the proposed experiments determined the time course of changes in MHC mRNA

during 6 to 18 hours of MV. To test our hypothesis we measured type I, IIa, IId/x, and IIb

MHC mRNA content in diaphragms from control, SB, and MV animals after 6, 12, and

18 hours of SB or MV. The measurements were made using a portion of the costal

diaphragm from the same group of animals used to test the first hypothesis. The

experimental design, surgical procedures, care, diaphragm removal, necropsy

examination, and blood culture have been described in Specific Aim #1.

Methods Used: Biochemical Assays

Total RNA isolation

A portion of the costal diaphragm, -50 mg, was homogenized in 1.5 mL of Trizol

(Invitrogen, Carlsbad, CA) and processed according to the manufacture's instructions.

This protocol is based on the method described by Chomczynski and Sacchi (34). The

sample was centrifuged at 12,000 x g for 10 minutes to the remove insoluble material.

The RNA portion, the upper aqueous phase, was transferred and incubated at room

temperature for 5 minutes. The RNA was extracted with bromochloropropane,

precipitated with isopropanol, washed with 75% ethanol, and pelleted via centrifugation.









The pellet was resuspended in RNAse free water (Sigma, St. Louis, MO). The

concentration and purity of the total RNA extracted was measured spectrophotometrically

at 260 nm and at 280 nm in lx TE buffer (Promega, Madison, WI). Ideally, the ratio of

A260/A280 should be greater than 1.8. This is a measure of RNA purity. Absorbance at 260

nm (A260) reflects the RNA contration and absorbance at 280 nm (A280) reflects the

protein content. The concentration of RNA was determined via the equation:

OD at 260 x 40 1
ugRNA IpuL = x-
# of /L read 10

The optical density (OD) of RNA at 260 nm (OD260) is assumed to equal 40 [tg/[tL (178).

This method yields un-degraded RNA, free of DNA and proteins.

The integrity of the extracted total RNA was verified by gel electrophoresis of 1 |tg

RNA on an 1% agarose tri(hydroxymethyl)aminomethane (Tris)-borate-EDTA buffer

(TBE) gel containing ethidium bromide using lx TBE as the running buffer. Both the

28S and 18S ribosomall RNA) bands were clear and distinct in the intact samples.

Samples that did not demonstrate these characteristics (i.e., degraded samples) were

discarded and another tissue sample from the same animal was processed and an un-

degraded RNA sample was then analyzed. The total RNA samples were then stored at -

800C until analysis.

Reverse transcription (RT)

Total RNA was reverse transcribed and amplified via PCR for each diaphragm

sample. Briefly, 1 |tg of total RNA was reverse transcribed using SuperScript II RT

(Invitrogen, Carlsbad, CA) and a mix of oligo dT (100 ng/reaction) and random primers

(200 ng/reaction) in a final volume of 20 ptL according to the protocol provided by the









manufacturer. An equal number of samples from each group were included in each run.

The samples were then stored at -800C until used for PCR.

Polymerase chain reaction (PCR)

MHC content was determined via relative RT-PCR with 18S serving as the internal

standard (Ambion, Austin TX). The MHC primer sequences used have been published by

Dr. Ken Baldwin's group (178) and are shown in Table 3-1. The primer for the five prime

(5') end of each mRNA was designed from a highly conserved region in all known rat

MHC genes -600 base pairs upstream of the stop codon (99). The four adult rat MHC

isoforms (I, IIa, IId/x, and IIb) are identical in this region. This allowed for the design of

a "common" primer with the following sequence:5' GAA GGC CAA GAA GGC CAT C

3' (178). The primers for the three prime (3') end were derived from the 3'untranslated

regions (UTR) of each of the different MHC genes, the sequences for each rat MHC gene

are highly specific in this region (42, 65, 66).

In order to account for differences in the initial amount of total RNA and to serve

as an internal standard, 18S ribosomal RNA was co-amplified with the target cDNA

(mRNA) in each PCR sample. The Alternate 18S Internal Standard kit (Ambion, Austin,

TX) was used. The 18S primers were mixed with the provided competimer in a 1:4 ratio.

The 18S competimer is required to decrease the 18S signal. The 18S primer to

competimer ratio was optimized such that amplification of the target cDNA and 18S

ribosomal RNA was similar (Ambion, Relative RT-PCR kit protocol).

The PCR conditions were as follows: 2 mM MgC12 in standard PCR buffer

(Invitrogen, Carlsbad, CA), 0.2 mM dNTP, 0.2 [tM of the common primer, 0.2 [tM of

one of the four gene specific primers, 0.5 [tM 18S primer/competimer mix, 2 p.L of the









diluted RT product (1 pIL of each RT reaction was diluted 40 fold before PCR

amplification), and 0.75 units of Taq polymerase (Invitrogen, Carlsbad, CA) in a final

volume of 25 ILL. All four MHC genes were amplified in four separate reactions for each

experimental group and one sample form each experimental group was included in each

PCR run. PCR was carried out with an initial 3 minute denaturation step at 960C,

followed by 24 cycles, each cycle consisting of 45 seconds at 960C (denaturation), 60

seconds at 500C (primer annealing), 90 seconds at 720C (extension), and a final step of 3

minutes at 720C using the Stratagene Robocycler. The number of cycles was determined

to be on the linear portion of a semilog plot of the yield (see below) expressed as a

function of cycle number. The PCR products were separated by agarose gel

electrophoresis [20 pIL sample of the PCR product loaded on 2.0% agarose gels (in lx

TBE buffer) containing 0.2 [tg/mL ethidium bromide] for visualization of the PCR

products.

Analysis of gels

A digital image of each gel was captured and the bands were analyzed via

computerized densitometry (Gel-Doc 2000, Bio-Rad, Hercules, CA). The integrated areas

of each target (MHC) and 18S internal control fragment DNA band were determined with

the local background subtracted. The integrated area of the target band was normalized to

the integrated area of the corresponding 18S internal control fragment, thus correcting for

any differences in PCR reaction efficiencies. The value for each MHC mRNA is

expressed as MHC mRNA/18S.









Statistical analysis

Planned Comparisons were made between relevant groups with a Bonferroni

correction for the number of comparisons conducted. Significance was established at

p < 0.05.

Table 3-1. Oligonucleotide primers used for the PCR reactions
MRNA Common Primer Antisense Primer Sample
(5' end) (3' end) cDNA


Type I MHC


Type IIa MHC

Type IId/x MHC

Type IIb MHC


5' GAA GGC CAA
GAA GGC CAT C 3'
5' GAA GGC CAA
GAA GGC CAT C 3'
5' GAA GGC CAA
GAA GGC CAT C 3'
5' GAA GGC CAA
GAA GGC CAT C 3'


5' GGT CTC AGG
GCT TCA CAG GC 3'
5' TCT ACA GCA
TCA GAG CTG CC 3'
5' GGT CAC TTT
CCT GCT TTG GA 3'
5' GTG TGA TTT
CTT CTG TCA CC 3'


Sample cDNA is the size of the myosin heavy chain (MHC) mRNA PCR product in base
pairs (bp).



common stop codon
primer
region -600 bp MHC sequences 3'UTR





Figure 3-2. Schematic representation of the myosin heavy chain (MHC) genes, the
common primer is identical in all sequences, followed by -600 base pairs (bp)
of coding sequence. A stop codon and 3'-untranslated region (UTR) that are
highly specific for each MHC gene with little or no sequence similarity
among gene family members are depicted.


596 bp

570 bp

574 bp

590 bp














CHAPTER 4
RESULTS

Morphological, Physiological, and Post Mortem Observations

The body mass characteristics of each experimental group are presented in Table 4-

1. No significant differences (p > 0.05) existed in pre-experiment or post-experiment

body mass between groups. Importantly, no group experienced a significant (p > 0.05)

loss of body mass over the course of the experiment, indicating adequate hydration

during the experimental period. Additionally, all animals urinated and experienced

intestinal transit during the experimental period.

Heart rate and systolic blood pressure were monitored as a means of determining

animal homeostasis during the experimental period. The mean heart rate and systolic

blood pressure data are presented in Tables 4-2 and 4-3, respectively. Heart rate and

blood pressure were within normal ranges and were well maintained during the

experiments. Further, note that the heart rate and blood pressure responses were very

similar between the SB animals and the MV animals at each time point.

Post mortem examination of the SB and MV animals included a necropsy and

blood culture. No animals demonstrated any signs of infection, weight loss, or post

mortem abnormalities, and all blood cultures were negative for bacteria. Additionally, the

colonic temperature of each animal remained constant, 37 + 0.5C, during the

experiments. Collectively, these results indicate that our aseptic surgical technique

successfully prevented infection.









Influence of Mechanical Ventilation on Protein Synthesis

MV resulted in depressed protein synthesis in the diaphragm within the first 6

hours; this reduced rate of protein synthesis persisted throughout the remainder of the

experimental period. Note that within the first 6 hours, the rate of both mixed muscle

protein (MMP) (-30%) and MHC (-65%) protein synthesis significantly decreased

(p < 0.05). Precursor pool 13C enrichment, MMP and MHC 13C enrichment, and MMP

and MHC fractional synthetic rates are each presented in Figures 4-1, 4-2 and 4-3.

Enrichment values are expressed as mole percent in excess (MPE). Mole percent in

excess is the enrichment of 13C above natural levels. Plasma enrichment of 13C in MPE

was determined from the baseline plasma sample in each animal before infusion. Tissue

fluid, MMP, and MHC 13C enrichment in MPE was calculated using the acute anesthesia

animals as the baseline measure of naturally occurring levels of 13C.

Figure 4-1 reports plasma [13C]leucine and [1-13C]ketoisocaproic acid ([13C]KIC)

enrichment. The plateau in the enrichment of the plasma precursor pools indicates that a

steady-state was achieved by the 6th hour of infusion. No significant differences

(p > 0.05) existed in the enrichment of plasma [13C]leucine or [13C]KIC after 5 hours of

infusion compared to 6 hours of infusion within groups (e.g., SB 6 at hour 5 vs. hour 6).

Additionally, the enrichment of plasma [13C]leucine or [13C]KIC did not differ between

groups (e.g., SB 6 vs. MV 6). Rates of protein synthesis calculated using the plasma

[13C]leucine and [13C]KIC precursor pools were made using the 6 hour enrichment

values.

The [13C]leucine enrichment of the tissue fluid precursor pool did not differ

between time matched groups (Figure 4-2). Note that the 18 hour SB and MV groups









each experienced significantly greater (p < 0.05) tissue fluid [13C]leucine enrichment than

did their 6 and 12 hour counterparts.

By averaging the endogenous amount of 13C in the diaphragms from the Control

acute anesthesia group and subtracting the amount of endogenous 13C from all SB and

MV values we derived the degree of 13C enrichment of the diaphragm proteins. The

difference between the endogenous 13C content and the measured 13C after [13C]leucine

infusion is termed the degree of enrichment.

The SB group experienced a significant (20%, p < 0.05) decrease in diaphragmatic

MMP [13C]leucine enrichment over time (hour 6 to hourl8). In comparison to their time

matched counterparts, the MV animals experienced a significant (30-34%, p < 0.05)

decrease in diaphragmatic MMP [13C]leucine enrichment (Figure 4-3). [13C]leucine

enrichment of MHC protein in the diaphragm remained constant over time in the SB

group. A significant (68 to 75%, p < 0.05) decrease in the enrichment of diaphragm MHC

protein was observed during MV at each time point (Figure 4-3).

After measurement of precursor pool (plasma and tissue fluid) and MMP and MHC

enrichment, calculation of the fractional synthetic rate of protein synthesis was

performed. The fractional synthetic rate of both MMP and MHC protein was calculated

using all three surrogates of [13C]leucy-tRNA: plasma [13C]leucine, plasma [13C]KIC, and

tissue fluid [13C]leucine. MV significantly (p < 0.05) slowed the fractional synthetic rate

of both MMP and MHC protein synthesis (Figure 4-4, Figure 4-5, and Table 4-4).

Regardless of which precursor pool was used for these calculations the decreased

fractional synthetic rate remained. However, for clarity, only the fractional synthetic rate









calculated using the tissue fluid [13C]leucine precursor pool is presented in graphic form

(Figure 4-4 and Figure 4-5).

Mixed muscle protein synthesis, a measure of whole muscle protein synthesis,

slowed significantly (30%, p < 0.05) during the first 6 hours of MV as compared to the

time matched SB 6 group (Figure 4-4 and Table 4-4). The 30% decrease in the rate of

MMP synthesis persisted at hour 12 (MV 12 compared to SB 12, -26%) and hour 18 (MV

18 compared to SB 18, -29%).

Myosin heavy chain protein synthesis was measured in order to estimate the impact

of MV of the rate of contractile protein synthesis. Within the first 6 hours of MV, MHC

protein synthesis slowed significantly (66%, p < 0.05) as compared to the time matched

SB 6 group (Figure 4-5 and Table 4-4). In parallel with the MMP synthesis rates, the

decrease in MHC protein synthesis rates after MV remained constant as compared to

each time matched SB group.

Total RNA and Myosin Heavy Chain mRNA in the Diaphragm after Spontaneous
Breathing and Mechanical Ventilation

Total RNA is -80-85% ribosomal RNA (rRNA) and can be used as an index of the

quantity of ribosomal subunits and as an indirect index of the synthetic capacity of the

tissue. In contrast, mRNA constitutes -2-3% of the total RNA pool. Total RNA was

isolated from each diaphragm and the mRNA encoding the four adult MHC phenotypes

was then measured to determine if the observed decrease in protein synthesis was due, in

part, to a decrease in total RNA and/or MHC mRNA. The total RNA findings are

presented in Table 4-5. Exposure to the anesthetic (SB groups) or MV did not affect the

amount of total RNA isolated from the diaphragms of any of the groups.









Once isolated from the diaphragm, total RNA was reverse transcribed. The cDNA

and the 18S rRNA control fragment were then amplified via PCR and the products were

separated electrophoretically on 2% agarose gels stained with ethidium bromide. An

example of the products is depicted in Figure 4-6. In each lane (top to bottom) the two

products, the amplified target mRNA and the amplified 18S internal standard fragment,

were present. The upper band is the amplified target mRNA and the lower band is the

18S rRNA internal standard fragment. The expected size of each amplified target mRNA

product is 596 bp, 570 bp, 574 bp, 590 bp for type I, IIa, IIx, and lib MHC, respectively.

The expected 18S control fragment size is 324 bp.

Figure 4-7 through Figure 4-10 shows MHC mRNA results. The MHC mRNA data

are expressed relative to the 18S rRNA internal standard product to account for variations

in the RT-PCR process. Exposure to prolonged anesthesia (SB) or MV did not alter the

relative amount of any of the four MHC mRNA's. The graphic results for each MHC

mRNA are depicted in Figure 4-7 through Figure 4-10.



Table 4-1. Animal body mass before and after experimental period
Group Body Mass (g) before Body Mass (g) after
Control 255.0 + 6.8 ----
SB 6 251.9 3.6 251.7 3.6
MV 6 248.5 + 4.2 248.5 4.2
SB 12 259.7 + 3.0 258.3 + 3.0
MV 12 262.8 + 2.5 262.7 2.3
SB 18 259.0 + 3.8 259.4 4.2
MV 18 259.7 + 3.1 259.4 + 3.3
Values are means standard error (SE) with n = 10 per group. SB = spontaneously
breathing. MV = mechanically ventilated.









Table 4-2. Heart rate response during MV and SB
Group Time zero 6th hour 12th hour 18th hour
Control N/D

SB 6 343 9 352 6

MV 6 357 3 360 6

SB 12 351 5 361 1 350 10

MV 12 375 7 358 14 364 + 6

SB 18 340 12 360 11 324 10 322 22

MV 18 374 10 363 5 388 11 395 17
Values are means SE expressed in beats per minute with n = 10 per group. N/D = no
data. SB = spontaneously breathing. MV = mechanically ventilated.

Table 4-3. Systolic blood pressure response during MV and SB
Group Time zero 6th hour 12th hour 18th hour
Control N/D

SB 6 135 4 109 5

MV 6 122 3 108 5

SB 12 137 3 129 6 103 7

MV 12 154 6 107 11 106 8

SB 18 128 5 112 5 96 5 92 3

MV 18 138 3 110 5 100 8 98 7
Values are means SE expressed in mmHg with n = 10 per group. N/D = no data. SB =
spontaneously breathing. MV = mechanically ventilated.
















13C-Leucine




13C-KIC


-*- SB 6
-0- MV6
-i- SB 12
-v- MV12
-U- SB 18
-0- MV18


Tracer Infusion Time (hr)
Plasma [13C]leucine and plasma [13C]ketoisocaproic acid ([13C]KIC)
enrichment. Values are means SE expressed in mole percent in excess of
natural 13C abundance with n = 10 per group. SB = spontaneously breathing
animals; MV = mechanically ventilated animals. Hour 5 = plasma
[13C]leucine or [13C]KIC after 5 hours of infusion; hour 6 = plasma
[13C]leucine or [13C]KIC after 6 hours of infusion.


25



20 -


u)
0 o
X
E0
CL


15



10


Figure 4-1.


a






61


16

14

12 -1

E e T *
10




o 6

4

2

0
SB 6 MV 6 SB 12 MV12 SB 18 MV18

Figure 4-2. Tissue fluid [13C]leucine enrichment in the diaphragm. Values are means +
SE expressed in mole percent in excess of natural 13C abundance measured in
Control animal diaphragm with n=10 per group. SB = spontaneously
breathing animals; MV = mechanically ventilated animals. $ Significantly
different (p < 0.05) from SB 18; significantly different (p < 0.05) from MV
18.








0.18

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02


9


ewe


MIVP
: 1 HC


-I-


0.00 --


5


A
1t


SB6 IW6 SB12 IW12 SB18 IW18

Figure 4-3. Mixed muscle protein and myosin heavy chain [13C]leucine enrichment in the
diaphragm. Values are means + SE expressed in mole percent in excess of
natural 13C abundance measured in Control animal diaphragm with n=10 per
group. MMP= mixed muscle protein; MHC = myosin heavy chain; SB =
spontaneously breathing animals; MV = mechanically ventilated animals. *
Significantly different (p < 0.05) from time matched SB; t significantly
different (p < 0.05) from SB 6, t significantly different (p < 0.05) from MV


--7,]









0.30


0.25


0.20 -
T :1:
T
0.15


0.10


0.05


0.00
SB 6 MV6 SB 12 MV12 SB 18 MV18


Figure 4-4. Fractional synthetic rates of mixed muscle protein (MMP) by calculation with
tissue fluid [13C]leucine as the surrogate measure of the [13C]leucyl-tRNA
precursor pool. Values are means + SE expressed in percent per hour (%/hr)
with n=10 per group. SB = spontaneously breathing animals; MV =
mechanically ventilated animals. Significantly different (p<0.05) from time
matched SB group; significantly different (p < 0.05) from SB6.









0.14


0.12


0.10

0.08


0.06


0.04


0.02


0.00


Figure 4-5.


Fractional synthetic rates of myosin heavy chain (MHC) protein by
calculation with tissue fluid [13C]leucine as the surrogate measure of the
[13C]leucyl-tRNA precursor pool. Values are means + SE expressed in
percent per hour (%/hr) with n=10 per group. SB = spontaneously breathing
animals; MV = mechanically ventilated animals. Significantly different
(p<0.05) from time matched SB group; significantly different (p < 0.05)
from SB 6.


S6 V6 S 12 V12 S18 V18


SB 6 MV 6 SB12 MV12 SB18 MV18









Table 4-4. Fractional synthetic rates of mixed muscle protein and myosin heavy chain
protein by calculation with each surrogate of the [13C]leucyl-tRNA precursor
pool.


Group

6 hour SB

6 hour MV

12 hour SB

12 hour MV

18 hour SB

18 hour MV


Plasma [13C]leucine (%/hr)
MMP MHC

0.1381 + 0.0077 0.0514 + 0.0068

0.0886 + 0.0077 0.0178 0.0056 *

0.1013 0.0044 8 0.0487 + 0.0088

0.0624 0.0066 t 0.0173 0.0104 *

0.1174 0.0055 8 0.0419 0.0056

0.0813 0.0055 0.0118 0.0027 *


Plasma [13C]KIC (%/hr)


Group

6 hour SB

6 hour MV

12 hour SB

12 hour MV

18 hour SB

18 hour MV


MMP

0.2454 + 0.0127

0.1747 0.0110 *

0.1827 0.0077

0.1281 + 0.0110 t

0.2277 + 0.0110

0.1466 0.0110*


MHC

0.0920 + 0.0132

0.0321 + 0.0088 *

0.0864 + 0.0121

0.0304 + 0.0176*

0.0774 + 0.0099

0.0225 + 0.0051*









Table 4-4. (continued)
Tissue Fluid [13C]leucine (%/hr)
Group MMP MHC
6 hour SB 0.2040 + 0.0110 0.0990 + 0.0180
6 hour MV 0.1691 0.0110 0.0341 + 0.0092*
12 hour SB 0.1879 0.0088 8 0.0899 + 0.0165
12 hour MV 0.1388 0.0088 t 0.0310 + 0.01650*
18 hour SB 0.1636 0.0077 0.0584 0.0048
18 hour MV 0.1164 0.0055 t 0.0187 0.0044*
Values are means SE expressed in percent per hour with n= 10 per group. MMP =
mixed muscle protein; MHC = myosin heavy chain; SB = spontaneously breathing
animals; MV = mechanically ventilated animals. Significantly different (p<0.05) from
time matched SB group; significantly different (p < 0.05) from SB 6, t significantly
different (p < 0.05) from MV 6. Note that the Tissue Fluid [13C]Leucine data are also
presented in Figures 4-4 and 4-5.


Table 4-5. Total RNA obtained from the costal diaphragm
Group Total RNA ([tg/mg)
Control 0.923 0.200
6 hour SB 0.937 0.032
6 hour MV 0.962 0.022
12 hour SB 0.898 0.017
12 hour MV 0.910 + 0.018
18 hour SB 0.959 0.028
18 hour MV 0.991 + 0.023
Values are means SE expressed as |tg of total RNA and as the amount ([tg) of total
RNA per mg wet weight of diaphragm with n = 10 per group. SB = spontaneously
breathing; MV = mechanically ventilated.










MWM
(BP)
60C0
300O


Figure 4-6. RT-PCR products separated on a 2% agarose gel with n=10 per group. C =
Control, acute anesthesia animals; SB = spontaneously breathing animals;
MV = mechanically ventilated animals; MWM = molecular weight marker in
basepairs (bp); T = target MHC mRNA; 18S = 18S rRNA internal standard.


0 I I I I I I I
Control SB 6 MV6 SB 12 MV12 SB 18 MV18

Figure 4-7. Relative type I MHC expression. Values are means + SE expressed as type I
MHC mRNA normalized to 18S rRNA internal standard (MHC mRNA / 18S)
with n=10 per group. C = Control, acute anesthesia animals; SB =
spontaneously breathing animals; MV = mechanically ventilated animals.


Ila

" SB6 MV6 SB12 MV12 SB18 MV18 C SB6 MV6 B172 MV12 SB18 MV18

IIX Ilb

" SB MV6 SB12 MV12 ;ii18 MV18 C SB6 MV6' SB12 MV12 SB18 MV18


_L






























Control SB 6 MV 6 SB 12 MV 12 SB 18 MV 18


Figure 4-8. Relative type IIa MHC expression. Values are means + SE expressed as type
IIa MHC mRNA normalized to 18S rRNA internal standard (MHC mRNA /
18S) with n=10 per group. C = Control, acute anesthesia animals; SB =
spontaneously breathing animals; MV = mechanically ventilated animals.














CT 2
CO







C1-




0



Control SB 6 MV 6 SB 12 MV12 SB 18 MV 18

Figure 4-9. Relative type IIx MHC expression. Values are means + SE expressed as type
IIx MHC mRNA normalized to 18S rRNA internal standard (MHC mRNA /
18S) with n=10 per group. C = Control, acute anesthesia animals; SB =
spontaneously breathing animals; MV = mechanically ventilated animals.










2




oo


z


I









0
Control SB 6 MV 6 SB 12 MV12 SB 18 MV18

Figure 4-10. Relative type IIb MHC expression. Values are means + SE expressed as type
lib MHC mRNA normalized to 18S rRNA internal standard (MHC mRNA /
18S) with n=10 per group. C = Control, acute anesthesia animals; SB =
spontaneously breathing animals; MV = mechanically ventilated animals.














CHAPTER 5
DISCUSSION

Overview of Principle Findings

These experiments investigated the affect of 6 to 18 hours of MV on protein

synthesis and MHC mRNA in the rat diaphragm. Our results support the hypothesis that

the MV-induced diaphragmatic atrophy is, at least in part, due to a decreased rate of total

(MMP) and myosin heavy chain (MHC) protein synthesis. Indeed, within the first 6 hours

of MV MMP synthesis decreased by -30% and MHC protein synthesis decreased by

-65%. These decrements in protein synthesis persisted throughout the 18 hours of MV.

In contrast, our data do not support the postulate that MV alters pretranslational events in

the diaphragm as indicated by the observation that MV did not alter MHC mRNA

content. A detailed discussion of these points follows.

Impact of Mechanical Ventilation on Protein Synthesis in the Diaphragm

Mixed Muscle Protein Synthesis

MMP synthesis is the average synthetic rate of all proteins (e.g., contractile

proteins, sarcoplasmic reticulum proteins, enzymatic proteins) in the muscle sample and

was measured as an index of total protein anabolism in the diaphragm. The fractional rate

of MMP synthesis in the diaphragm was measured after three periods of MV (6, 12, and

18 hours), and compared to time matched controls. The SB 6 MMP synthetic rate

calculated using tissue fluid [13C]leucine was -0.2 %/h. These results are lower than

published MMP synthetic rates in the rat diaphragm, -0.4%/h to 0.6%/h (50, 127, 169). It

should be noted that young (-100g) rapidly growing animals were used in these previous









studies and this may have contributed to the differences in the rates. However, our

measured protein synthetic rates are similar to those measured in other adult rat skeletal

muscles (e.g., 0.16%/h in quadriceps (16), 0.23%/h in gastrocnemius (28), and 0.33%/h

in soleus (162)). Hence, the measured rates of MMP synthesis in the rat diaphragm in the

current study are consistent with rates reported in the literature for adult animals.

The MV-induced 30% decrease in MMP synthesis in the diaphragm occurred

during the first 6 hours of MV. Additionally, the MV-induced decrement in protein

synthesis remained steady after 12 and 18 hours of MV. This rapid change in skeletal

muscle protein synthesis has also been observed during periods of inactivity in rat

gastrocnemius. Indeed, 6 hours of hindlimb immobilization leads to a 37% decrease in

MMP synthesis (28). Similarly, the observed decrease in MMP synthesis in immobilized

rat gastrocnemius remains at this level after 2 days of immobilization (168). Therefore,

the observed decrease in MMP synthesis after 12 and 18 hours of MV in the rat

diaphragm is consistent with the rat hindlimb immobilization data (168) and indicates

that the diaphragm, like other skeletal muscles, is sensitive to loading state. Once

unloaded, via MV, protein synthesis in the diaphragm rapidly decreases and a new

steady-state of protein synthesis is established.

Myosin Heavy Chain Protein Synthesis

MHC is an essential component of the contractile apparatus and constitutes -25%

of skeletal muscle mass (122, 182). Importantly, force generation is proportional to the

amount of myofibrillar protein within the fiber and thus a decrease in the rate of MHC

synthesis would lead to a decrease in the force generating ability of the diaphragm (17,

26, 125). Accordingly, the rate of MHC protein synthesis was measured after 6, 12, and

18 hours of MV and compared to time matched controls.









These were the first experiments to measure the rate of MHC protein synthesis in

the rat diaphragm. The rate of protein synthesis calculated using tissue fluid [13C]leucine

is -0.1%/h in contracting diaphragm (SB 6). Previous investigations report MHC protein

synthesis rates of 0.1%/h in the rat quadriceps (16) and -0.25%/h in the rat soleus (162).

It is unclear why the rates in the diaphragm are not in line with those of the more active

soleus.

The observation that MHC protein synthesis rate is slower than the MMP synthesis

rate suggests that MHC protein turns over slower (longer half-life) than other proteins in

the MMP pool. The rates of MHC protein synthesis in the current study are -60% less

than the MMP synthesis rate. Previous studies report an -40% difference in quadriceps

(16) and an -25% difference in soleus (162). The data in the current study suggest that

the MHC protein pool in the diaphragm is turning over slower than MHC protein in other

skeletal muscles.

Six hours of MV resulted in an -65% decrease in the rate of MHC protein

synthesis. This decrease in MHC protein synthesis was maintained through 18 hours of

MV. This rapid decrease in the rate of diaphragmatic MHC protein synthesis was more

severe than the reported change after 5 hours of hindlimb unloading where a consistent

but non-significant decrease was measured in the soleus (162). A possible explanation for

the divergent findings is that during MV the diaphragm is not contracting but the soleus

is free to contract (against little resistance) during hindlimb unloading. Hence, this level

of activation in the soleus may serve to attenuate the decrease in protein synthesis during

hindlimb unloading compared to MV. The change in MHC protein synthesis has not been

measured during hindlimb immobilization but the synthetic rate of another essential









contractile protein, a-actin, has been examined (173). During the first 6 hours of

hindlimb immobilization, a-actin protein synthesis in the rat gastrocnemius decreases

-66% (173). The MHC protein synthesis data in the current study and the a-actin protein

synthesis data after 6 hours of hindlimb immobilization (173) indicate that skeletal

muscle rapidly adapts to unloading by significantly decreasing the rate of contractile

protein synthesis.

Myosin Heavy Chain mRNA

The fractional synthetic rate of specific proteins can be altered by pretranslational

events leading to a decrease in the amount of a given mRNA (e.g., the rate of

transcription or turnover of MHC mRNA). As discussed previously, MV significantly

slows both MMP and MHC protein synthesis. Because MHC is the most predominant

protein in skeletal muscle it was hypothesized that 6 to 18 hours of MV would alter

pretranslational events in the diaphragm (i.e., MHC mRNA content would decrease). The

data, however, do not support this hypothesis.

As indicated in Figures 4-7 through 4-10, 6 to 18 hours of MV did not alter MHC

mRNA content in the diaphragm. These observations are consistent with previous studies

of locomotor skeletal muscle disuse. In the rat soleus 3-MHC (slow) mRNA content does

not significantly change after 5 hours or 7 days of hindlimb unloading, yet the synthetic

rate of MHC protein decreases significantly (162). Further, 6 to 72 hours of hindlimb

immobilization does not change the amount of a-actin mRNA in the rat gastrocnemius

but the synthetic rate of a-actin protein was decreased -65% during the first 6 hours

(173). The data from the current study in conjunction with previous studies demonstrates

that the rapid decrease in the rate of protein synthesis in the diaphragm, like other skeletal









muscles, is not due to a change in MHC mRNA content. Nonetheless, a recent report

indicates that extended periods of MV, >48 hours, does alter MHC mRNA (180). Using

Northern blot analysis, this group reported that >48 hours of MV increases MHC IIa

(70%) and MHC IIx (22%) mRNA, with little change in lib MHC mRNA (4%), and no

change in type I mRNA (180). Immunohistochemistry was used to fiber type a portion of

diaphragm after >48 hours of MV and detected a significant decrease in type I fibers and

a significant increase in fibers co-expressing both type I and II MHC protein (180). These

findings (180), in conjunction with the present study, suggest that during the first 18

hours of MV there is no measurable change in MHC mRNA but over the course of the

next 36 hours MHC mRNA expression does change and leads to a slow-to-fast MHC

shift associated with skeletal muscle unloading.

Regulation of Protein Synthesis

Protein synthesis is the culmination of many events, including transcription and

translation; all of which are highly regulated. The rapid decrease in protein synthesis after

MV could be due to the inhibition of one or both of these steps. A discussion of key

points of regulation of protein synthesis as they pertain to the MV-induced decrease in

protein synthesis follows.

Transcription

In healthy active skeletal muscle, MHC protein expression appears to be regulated

by transcriptional events (18). For example, 3-MHC promoter region activity in the

soleus is significantly decreased after 7 days of inactivity (58, 80). Additionally, changes

in mRNA expression precede changes in protein expression measured from the 4th day to

the 90th day of inactivity (79). Thus, over a period of days/weeks/months MHC protein









expression is regulated by transcriptional events but during the first hours of reduced use

(e.g., 18 hours) protein expression is regulated by post-transcriptional events.

MV did not change the amount of total RNA (Table 4-5) or MHC mRNA (Figures

4-7 through 4-10). Hence, the observed decrease in the rate of protein synthesis without a

decrease in total RNA and MHC mRNA is indicative of a decrease in translational

efficiency (the amount of MHC protein synthesized per amount of MHC mRNA). A

decrease in translational efficiency occurs when one or more steps of translation is

hindered.

Translation Initiation

The process of translating mRNA into a nascent polypeptide chain includes

initiation, elongation, and termination. In the following sections control of initiation will

be discussed in terms of relevant protein (initiation) factors and the pathway that controls

the assembly of the initiation complex. This will be followed by a discussion focusing on

the regulation of elongation and termination via the 3' end of mRNA.

Translation initiation is the result of a series of steps culminating in the 40S and

60S ribosomal subunits binding to mRNA. The regulatory processes involved in initiation

have been well elucidated. Specifically, proteins known as eukaryotic initiation factors

(elFs) are required for the engagement of the 40S ribosomal subunit and the 60S

ribosomal subunit with mRNA. elF function/activity is regulated by specific kinases and

phosphatases. Of particular interest is the regulation of eIF4E by 4E-binding protein

(BP)1. eIF4e is a critical component of the initiation complex and when bound by 4E-

BP1 initiation is hindered. 4E-BP1 binding of eIF4E is regulated by Akt (also known as

protein kinase B) and the putative kinase, mammalian target of rapammycin (mTOR)









(144). Akt and mTOR are central components of the Akt/mTOR pathway, which is

modulated by the loading state of skeletal muscle.

Akt acts directly on mTOR and the activity of Akt is sensitive to disuse. After 2

weeks of hindlimb suspension Akt protein expression and phosphorylated Akt (active

Akt) is significantly decreased (25). mTOR phosphorylation is modulated by Akt and 2

weeks of hindlimb suspension leads to a 60% decrease in mTOR phosphorylation (131).

Further, 2 weeks of hindlimb unloading increases 4E-BP 1 binding to eIF4E by >100%

(25). The results ofBodine et al. (25) and Reynolds et al. (131) suggest that hindlimb

unloading decreases the amount of Akt which in turn decreases mTOR activity leading to

increased 4E-BP1 binding to eIF4E (25) and thus preventing eIF4E from participating in

initiation. This is significant because it directly implicates the Akt/mTOR pathway in

muscle atrophy by inhibiting initiation.

In addition to controlling the phosphorylation state of 4E-BP 1, the Akt/mTOR

pathway controls the activity of the 70-kDa 40S ribosomal protein S6 kinase (p70S6k),

possibly through mTOR and directly by protein dependent kinase 1 (PDK1) (144). After

the phosphorylation of p70S6k its activity increases. Control of p70S6k is important

because this kinase controls the function of the ribosomal protein S6, a component of the

40S ribosomal subunit that is involved in tRNA recognition (144). After 12 hours of

hindlimb unloading or after 12 hours of denervation the phosphorylation state of p70S6k is

decreased 3-fold and remains depressed after 7 days (76). The decreased

phosphorylation state of p70s6k has also been reported after 2 weeks of hindlimb

suspension (25). Future experiments should explore the effect of MV on inhibition of

translation initiation in the diaphragm.









Translation Elongation and Termination

In addition to impairing initiation, reduced use of skeletal muscle impairs

elongation and termination. Previous studies (12, 88) indicate that during the initial hours

of reduced use protein synthesis in skeletal muscle stalls during elongation. Polysome

density is a measure of the number of ribosomes engaged in elongation per mRNA;

increasing in the number of ribosomes per mRNA increases the density of polysomes. Ku

and Thomason (88) studied a-actin polysome density after 18 hours of hindlimb

unloading and found a significant increase in polysome density. These data indicate that

assembly of the ribosomes on mRNA, (i.e., initiation) continues during the first 18 hours

of unloading. Further, increased polysome density indicates that elongation is somehow

inhibited.

As a follow up to the study of Ku and Thomason (88), Ashley and Russell (12)

tested the hypothesis that the 3' UTR of the j3-MHC regulates the decrease in protein

synthesis after 2 days of tenotomy in the rat soleus. Translation in the -3-MHC 3' UTR

was significantly decreased (12). Importantly, they found a significant increase in the

binding of a trans-acting protein factor in the 3' UTR (12). Based on these findings and

the work of Ku and Thomason (88) the authors suggest the following model: During

unloading the trans-acting protein binds to the 3-MHC mRNA 3' UTR with greater

affinity (12). This binding would physically prevent the ribosomes from reaching the stop

codon during elongation (i.e., translation would stall) (12). This would prevent a

completed protein from being released (termination) and thus repress protein expression

(12).









In summary, the MV-induced decrease in MMP and MHC protein synthesis in the

diaphragm may be due to alterations in the translational apparatus. Indeed, our finding

that MV does not alter diaphragmatic MHC mRNA levels but results in a decreased rate

of protein synthesis is consistent with this postulate. The current study did not measure

rates of initiation or elongation, inhibition of the Akt/mTOR pathway and/or ribosomal

stalling during elongation. However, one or more of these mechanisms may contribute to

the rapid decrease in protein synthesis induced by MV. This is an interesting area for

future research.

Critique of the Experimental Model

These experiments measured the changes in protein synthesis in the diaphragm

during the initial hours of MV. Due to the invasive nature of these experiments the rat

was used as the experimental model because of the biochemical and functional

similarities between the rat and human diaphragm. The rate of incorporation of the stable

tracer [13C]leucine into diaphragmatic proteins was used to measure protein synthesis. To

account for possible limitations of the experimental model a SB group was incorporated

into the experimental design. The SB animals underwent a surgical procedure identical to

the MV animals and received the same anesthetic for the same period of time. The SB

animals, therefore, served as time matched controls for the MV group so that any

alterations observed could be attributed to the effect MV. Of particular interest to these

experiments are the following considerations: the animals were not fed, the possibility

that anesthesia impacted protein synthesis, the length of the infusion period, and the

removal of blood.









Nutritional Status

The use of 13C[leucine] precluded feeding the MV and SB animals during the

experiments. 13C is a naturally occurring stable isotope present in all foods. Likewise,

leucine is a branched chain amino acid present in protein sources. Thus, feeding the

animals during the experiments would introduce an unknown amount of leucine with an

unknown amount of 13C and 12C into the animal's circulation and tissue (Kevin

Yarasheski, personal communication). This would dilute the 13C[leucine] tracer

administered to measure muscle protein synthesis by an unknown amount. Thus, the

interpretation of the 13C[leucine] enrichment data would be difficult if not impossible.

Therefore, to avoid confounding the 13C[leucine] enrichment measures, we chose not to

feed the MV and SB animals during the experimental period.

It was observed that protein synthesis decreased over time in the SB group.

Comparing the SB6 group to the SB 18 group the animals experienced a 32% decrease in

the rate of MMP synthesis and a 41% decrease in the rate of MHC synthesis. The

observed changes in the synthetic rate are consistent with the literature. Goldspink et al.

(62) report a 48% decrease in diaphragm MMP synthesis 23 hours post feeding and Bates

et al. (22) report a 44% decrease in the rate of limb-locomotor MHC synthesis 24 hours

post feeding. However, comparing the MV18 results to the time matched SB group a

29% decrease in MMP synthesis and a 68% decrease in MHC synthesis is still realized.

Therefore, the impact of MV on protein synthesis in the diaphragm was not obscured by

the nutrient status of the animals.

Anesthesia

The anesthetic agent, sodium pentobarbital, could have impacted protein synthesis

in the diaphragm. Nonetheless, rats anesthetized with 20 mg/kg sodium pentobarbital









(twice the dose used in the current experiments) for 1 hour did not experience a

significant decrease in protein synthesis in skeletal muscle (74). Additionally, general

anesthesia does not decrease protein synthesis in skeletal muscle in healthy humans

undergoing abdominal surgery (47). Collectively, these experiments indicate that protein

synthesis is not altered by anesthesia per se. The influence of continued exposure of any

given anesthetic agent (e.g., 18 hours) would be difficult to separate from the reduced use

during that state. However, the experiments reviewed above (47, 74) report normal rates

of protein synthesis in limb-locomotor skeletal muscle during periods of time that

reduced use would not be expected to have an affect on protein synthesis. These reports

(47, 74) indicate that anesthesia does not affect protein synthesis; therefore, the decreased

rate of protein synthesis in the diaphragm during MV is attributable to MV, not the

anesthetic.

Infusion period

Protein synthesis in the diaphragm was measured using the primed dose constant

infusion method with the stable tracer [13C]leucine. A plateau in 13C enrichment of the

plasma was achieved over the 6-hour infusion period (Figure 4-1). It is unknown if such a

plateau occurred within the tissue fluid of the diaphragm. The infusion period was 6

hours for all three experimental time points and tissue samples were taken at the

completion of the experimental period. Early work by Fern and Garlick (50)

demonstrated that a plateau in the enrichment of the plasma pool occurs within 2 hours

but enrichment of the labeled free amino acid in the tissue fluid of the diaphragm

continued to increase during 6 hours of infusion. The same authors (50) point out that if

the free amino acid pool in the plasma or tissue reflects the labeled protein precursor then

the calculated protein synthesis rate should be the same regardless of which precursor









pool is used in the calculation. Despite the continued rise in tissue fluid enrichment, the

authors (50) report similar rates of protein synthesis using each of the precursor pools,

with the plasma precursor pool giving the slowest rates. Similar to Fern and Garlick (50),

the rates of protein synthesis calculated using plasma [13C]leucine in the present

experiments underestimated the rates of protein synthesis. The concentration or

enrichment of [13C]leucine in the plasma should be greater than the enrichment of

"downstream" precursor pools such as tissue fluid and the transamination product of

leucine, KIC. Therefore, using plasma [13C]leucine as a precursor pool to calculate

protein synthesis should, and does, give a lower estimation of protein synthesis than

tissue fluid or KIC enrichment. The data in the present experiments indicate that plasma

[13C]KIC and tissue fluid [13C]leucine are appropriate surrogates of t-leucyl RNA.

Indeed, using these surrogates to calculate rates of protein synthesis yields similar rates

(Table 4-4).

The duration of the [13C]leucine infusion period was 6 hours. This duration was

chosen in order to achieve a plateau in [13C]leucine enrichment of the plasma precursor

pool (see Figure 4-1). Due to the length of the infusion period the rates of protein

synthesis are a rolling average of the 6-hour period. Thus, the rates of protein synthesis at

each time point may underestimate the actual rate of protein synthesis at any given

moment. This would be the most profound during the first 6 hours of MV, as the last hour

would be averaged in with the first.

Blood Removal

One milliliter of blood was drawn from each animal before [13C]leucine infusion

and after the 5th and 6th hours of infusion. Following each blood draw an equal volume of

normal saline was administered to prevent hypovolemia. The initial blood sample and the









sample taken after the 5th hour precluded our ability to monitor arterial blood gas during

the experiments. Nonetheless, we have demonstrated that our MV protocol results in only

minor disturbances in blood gas homeostasis over a 24-hour period (125). However, SB

animals typically experience some degree of respiratory acidosis without any significant

affect on diaphragmatic contractile function (125). During the present experiments we

relied on our previous experience with the mechanical ventilator and anesthesia

parameters and it is possible that blood gas homeostasis was not adequately maintained.

It should be noted that over the course of these experiments a high degree of surgical

success was achieved (i.e., 88% of the experiments were successful) suggesting that

animal homeostasis was well maintained despite our inability to monitor blood gas

homeostasis.

Mode of Mechanical Ventilation

Pressure-assist MV is commonly used to treat adult patients in intensive care units.

However, we used controlled MV for two reasons. First, because controlled MV results

in rapid diaphragmatic atrophy (167) the impact of controlled MV can be studied during

relatively short time periods. Second, controlled MV is clinically relevant as it is used in

adult patients following drug over dose, spinal cord injury and is commonly used in

certain pediatric situations (72).

Summary and Future Experiments

These experiments investigated the affect of MV on protein synthesis and MHC

mRNA in the rat diaphragm. The hypothesis that MV-induced diaphragmatic atrophy is,

at least in part, due to a decreased rate of total (MMP) and myofibrillar (MHC) protein

synthesis was supported. However, the hypothesis that MV alters pretranslational events

in the diaphragm was not supported.






84


Future experiments investigating the mechanisms that regulate protein synthesis in

the diaphragm during MV should follow several pathways. First, the role the Akt/mTOR

pathway plays in regulating the synthesis of diaphragmatic proteins during MV should be

studied. Secondly, polysome density of actin and myosin mRNAs after MV should be

measured to determine if these mRNAs are acutely regulated by arresting translation

during elongation. Additionally, identification of the trans-acting protein(s) binding to

the MHC mRNA 3' UTR should be pursued.















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